Specification of bilateral cardiac primordia and formation of the linear heart tube are highly conserved from Drosophila to humans. However, subsequent heart morphogenesis involving nonmesodermal neural crest cells was thought to be specific for vertebrates. Here, we provide evidence that a group of nonmesodermal cells that we have named heart-anchoring cells (HANCs) contribute to heart morphogenesis in Drosophila. We show that the homeobox genes ladybird (lb) known to be involved in diversification of cardiac precursors are expressed in HANCs and required for their specification. Interestingly, the HANCs selectively contact the anterior cardiac cells, which express lb as well. Direct interaction between HANCs and cardiac cells is assisted by a pair of cardiac outflow muscles (COMs), each of which selectively attaches to both the lb-expressing cardiac cells and HANCs. COM muscles seem to ensure ventral bending of the heart tip and together with HANCs determine the spatial positioning of the cardiac outflow region. Experimentally depleted cardiac lb expression leads to the disruption of the contact between the tip of the heart and either the COM muscles or the HANC cells, indicating a pivotal morphogenetic role for the lb expression within the heart. T he understanding of coordinated patterning of different cell types that build functional organs during embryogenesis seems to be an important challenge in developmental biology. Formation of the vertebrate heart provides an example of how the cells of mesodermal (heart primordia) and ectodermal (neural crest cells) origin may contribute to creating a fully functional organ (1). Interestingly, from the molecular and embryological points of view, the early stages of cardiogenesis, including specification of the heart primordia and formation of the beating heart tube, have been preserved from Drosophila to humans (2, 3). In this context, it becomes important to know whether nonmesodermal cells in Drosophila also contribute to a functional heart.The Drosophila heart (dorsal vessel) is a hemolymph-pumping organ that is frontally open and arranged in a repeat pattern of segmental units. At the end of cardiac morphogenesis, the posterior area of the dorsal vessel becomes enlarged and constitutes the definitive heart, whereas the anterior portion has a narrow diameter and is equivalent to the aorta. The heart is composed of two major mesodermal cell types: cardioblasts and pericardial cells (3, 4). The cardioblasts are aligned in two highly ordered rows that form the lumen of the heart. These cells are contractile and express a variety of muscle-specific proteins. The pericardial cells, whose function is unknown so far, are loosely associated with the cardioblasts and do not express any musclespecific marker. Recent studies of genes involved in heart development (5, 6) have revealed discrete subsets of cardioblasts and pericardial cells. According to the expression domains of heart identity genes, in each abdominal segment three types of cardioblasts can be detected, the a...