Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation.

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. We analyze here the formation of the Drosophila melanogaster cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. Our study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, we identify a genetic pathway in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data we propose a model for D. melanogaster cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. We suggest that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis.

Steroid-dependent modification of Hox function drives myocyte reprogramming in the Drosophila heart.

In the Drosophila larval cardiac tube, aorta and heart differentiation are controlled by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA), respectively. There is evidence that the cardiac tube undergoes extensive morphological and functional changes during metamorphosis to form the adult organ, but both the origin of adult cardiac tube myocytes and the underlying genetic control have not been established. Using in vivo time-lapse analysis, we show that the adult fruit fly cardiac tube is formed during metamorphosis by the reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation. We characterise the genetic control of the process, which is cell autonomously ensured by the modulation of Ubx expression and AbdA activity. Larval aorta myocytes are remodelled to differentiate into the functional adult heart, in a process that requires the regulation of Ubx expression. Conversely, the shape, polarity, function and molecular characteristics of the surviving larval contractile heart myocytes are profoundly transformed as these cells are reprogrammed to form the adult terminal chamber. This process is mediated by the regulation of AbdA protein function, which is successively required within these persisting myocytes for the acquisition of both larval and adult differentiated states. Importantly, AbdA specificity is switched at metamorphosis to induce a novel genetic program that leads to differentiation of the terminal chamber. Finally, the steroid hormone ecdysone controls cardiac tube remodelling by impinging on both the regulation of Ubx expression and the modification of AbdA function. Our results shed light on the genetic control of one in vivo occurring remodelling process, which involves a steroid-dependent modification of Hox expression and function.

[Genetic control of cardiac tube formation in Drosophila]. / Contrôle génétique de la formation du tube cardiaque chez la Drosophile.

In Drosophila, the heart is composed of a simple linear tube constituted of 52 pairs of myoendothelial cells which differentiate during embryogenesis to build up a functional mature organ. The cardiac tube is a contractile organ with autonomous muscular activity which functions as a hemolymph pump in an open circulatory circuit. The cardiac tube is organized in metamers which contain six pairs of cardioblasts per segment. Within each metamer the cardioblasts express a combination of genetic markers underlying their functional diversity. For example, the two most posterior cardiac cells in segments A5 to A7 differentiate into ostiae which allow the inflow of hemolymph in the tube. An additional axial information along the anteroposterior axis orchestrates the subdivision of the cardiac tube into an "aorta" in the anterior region and a "heart" in the posterior region which behave as distinct functional entities. The major pacemaker activity is located in the most caudal part of the heart. This analysis has being made possible by the identification and the utilization of specific morphological and genetic markers and an in vivo observation of cardiac function in the embryo. Functional organogenesis of the cardiac tube is accurately controlled by genetic programs that have been in part identified. Hox genes are responsible for the axial subdivision of the tube into functional modules. They activate, in their specific domains of expression, target genes effectors of the terminal differentiation. On the other hand, part of the information required for segmental information is provided by Hedgehog, a morphogen secreted by dorsal ectoderm, whose activity triggers the ostiae formation in the heart domain.

Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling.

The Drosophila larval cardiac tube is composed of 104 cardiomyocytes that exhibit genetic and functional diversity. The tube is divided into the aorta and the heart proper that encompass the anterior and posterior parts of the tube, respectively. Differentiation into aorta and heart cardiomyocytes takes place during embryogenesis. We have observed living embryos to correlate morphological changes occurring during the late phases of cardiogenesis with the acquisition of organ function, including functional inlets, or ostiae. Cardiac cells diversity originates in response to two types of spatial information such that cells differentiate according to their position, both within a segment and along the anteroposterior axis. Axial patterning is controlled by homeotic genes of the Bithorax Complex (BXC) which are regionally expressed within the cardiac tube in non-overlapping domains. Ultrabithorax (Ubx) is expressed in the aorta whereas abdominal A (abd-A) is expressed in the heart, with the exception of the four most posterior cardiac cells which express Abdominal B (Abd-B). Ubx and abd-A functions are required to confer an aorta or a heart identity on cardiomyocytes, respectively. The anterior limit of the expression domain of Ubx, abd-A and Abd-B is independent of the function of the other genes. In contrast, abd-A represses Ubx expression in the heart and ectopic overexpression of abd-A transforms aorta cells into heart cardiomyocytes. Taken together, these results support the idea that BXC homeotic genes in the cardiac tube conform to the posterior prevalence rule. The cardiac tube is also segmentally patterned and each metamere contains six pairs of cardioblasts that are genetically diverse. We show that the transcription of seven up (svp), which is expressed in the two most posterior pairs of cardioblasts in each segment, is dependent on hedgehog (hh) signaling from the dorsal ectoderm. In combination with the axial information furnished by abd-A, the segmental hh-dependent information leads to the differentiation of the six pairs of svp-expressing cells into functional ostiae.

Drosophila cardiac tube organogenesis requires multiple phases of Hox activity.

The segmented Drosophila linear cardiac tube originates from two cell lineages that give rise to the anterior aorta (AA) and the posterior cardiac tube. The three Hox genes of the Bithorax Complex as well as Antennapedia (Antp) have been shown to be expressed in the posterior cardiac tube, while no Hox gene is expressed in the anterior aorta. We show that the cells of the whole tube adopt the anterior aorta identity in the complete absence of Hox function. Conversely, ectopic expression of Antp, Ultrabithorax (Ubx), or abdominal-A (abd-A) transformed the anterior aorta into posterior cardiac tube by all available criteria, indicating an equivalent early function in their ability to direct a posterior cardiac tube lineage. We further demonstrate that Hox genes act in a subsequent step during cardiac tube organogenesis, specifically on the differentiation of posterior cardiac tube myocytes. In addition, while some of these functions are fulfilled equally well by any one of the three Hox genes, some others are specific to a given Hox. Notably, the gene encoding the anion transporter Na+-Driven Anion Exchanger 1 behaves as a Hox differential transcriptional target and is activated by abd-A in the heart and repressed by Ubx in the posterior aorta. This analysis illustrates the mechanisms by which Hox genes can orchestrate organogenesis and, in particular, allows a clear uncoupling of the different phases of Hox activity in this process.

Pericardin, a Drosophila type IV collagen-like protein is involved in the morphogenesis and maintenance of the heart epithelium during dorsal ectoderm closure.

The steps that lead to the formation of a single primitive heart tube are highly conserved in vertebrate and invertebrate embryos. Concerted migration of the two lateral cardiogenic regions of the mesoderm and endoderm (or ectoderm in invertebrates) is required for their fusion at the midline of the embryo. Morphogenetic signals are involved in this process and the extracellular matrix has been proposed to serve as a link between the two layers of cells. Pericardin (Prc), a novel Drosophila extracellular matrix protein is a good candidate to participate in heart tube formation. The protein has the hallmarks of a type IV collagen alpha-chain and is mainly expressed in the pericardial cells at the onset of dorsal closure. As dorsal closure progresses, Pericardin expression becomes concentrated at the basal surface of the cardioblasts and around the pericardial cells, in close proximity to the dorsal ectoderm. Pericardin is absent from the lumen of the dorsal vessel. Genetic evidence suggests that Prc promotes the proper migration and alignment of heart cells. Df(3)vin6 embryos, as well as embryos in which prc has been silenced via RNAi, exhibit similar and significant defects in the formation of the heart epithelium. In these embryos, the heart epithelium appears disorganized during its migration to the dorsal midline. By the end of embryonic development, cardial and pericardial cells are misaligned such that small clusters of both cell types appear in the heart; these clusters of cells are associated with holes in the walls of the heart. A prc transgene can partially rescue each of these phenotypes, suggesting that prc regulates these events. Our results support, for the first time, the function of a collagen-like protein in the coordinated migration of dorsal ectoderm and heart cells.