Specification of Drosophila Corpora Cardiaca Neuroendocrine Cells from Mesoderm Is Regulated by Notch Signaling

Kim SK (2011) Specification of Drosophila Corpora Cardiaca Neuroendocrine Cells from Mesoderm Is Regulated by Notch Signaling. PLoS Genet 7(8): e1002241. doi:10.1371/journal.pgen.1002241 Specification of Drosophila Corpora Cardiaca Neuroendocrine Cells from Mesoderm Is Regulated by Notch Signaling Sangbin Park 0 Erika L. Bustamante 0 Julie Antonova 0 Graeme W. McLean 0 Seung K. Kim 0 Norbert Perrimon, Harvard Medical School, Howard Hughes Medical Institute, United States of America 0 1 Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, United States of America, 2 Howard Hughes Medical Institute, Stanford, California, United States of America, 3 Department of Medicine (Oncology), Stanford University School of Medicine , Stanford, California , United States of America Drosophila neuroendocrine cells comprising the corpora cardiaca (CC) are essential for systemic glucose regulation and represent functional orthologues of vertebrate pancreatic a-cells. Although Drosophila CC cells have been regarded as developmental orthologues of pituitary gland, the genetic regulation of CC development is poorly understood. From a genetic screen, we identified multiple novel regulators of CC development, including Notch signaling factors. Our studies demonstrate that the disruption of Notch signaling can lead to the expansion of CC cells. Live imaging demonstrates localized emergence of extra precursor cells as the basis of CC expansion in Notch mutants. Contrary to a recent report, we unexpectedly found that CC cells originate from head mesoderm. We show that Tinman expression in head mesoderm is regulated by Notch signaling and that the combination of Daughterless and Tinman is sufficient for ectopic CC specification in mesoderm. Understanding the cellular, genetic, signaling, and transcriptional basis of CC cell specification and expansion should accelerate discovery of molecular mechanisms regulating ontogeny of organs that control metabolism. - Funding: The work was supported by the Howard Hughes Medical Institute. ELB was supported by NSRA pre-doctoral fellowship (5F31GM079930-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Recent work has revealed multiple features of evolutionary conservation in endocrine regulation of glucose metabolism. For example, in the fruit fly Drosophila melanogaster, insulin-producing cells (IPCs) in the brain and adipokinetic hormone-producing corpora cardiaca (CC) cells in the neuroendocrine ring gland are the respective functional orthologues of mammalian pancreatic bcells and a-cells [14]. Insect CC cells resemble neurons in multiple ways; CC cells are peptidergic secretory cells [5] that harbor dense core vesicles [6], and have axon-like projections to vascular, gut and brain targets [3,4,7]. Similar to pancreatic islet cells and neuronal cell subsets, CC cells also use KATP channels to regulate AKH secretion [3]. Targeted CC ablation results in marked hypoglycemia [3,4], demonstrating their role in glucose homeostasis. Thus, the molecular and physiological mechanisms governing CC endocrine function are strikingly similar to those of vertebrate pancreatic islets and neuroendocrine cells. Despite their crucial role in regulating systemic glucose balance, the embryonic origin of CC cells remains unclear. Based, in part, on their emergence near embryonic foregut, CC cells were initially proposed to originate from a placode in the foregut that produces the stomatogastric nervous system [8]. The CC cell anlage was later inferred to be the most anterior part of mesoderm, based on studies of gene expression in the embryonic head region [9,10]. Most recently, it was proposed that the CC cells originate from neuroectodermderived neuroblasts [11]. This latest study concluded that CC precursors originate from the same placode in which insulin producing neurons are born, and suggested that the developmental relationship between IPC and CC cells may be similar to that of hypothalamus and neuronal pituitary gland. Likewise, while a survey of candidate mutations revealed several genes required for CC development based on ontogenic similarities to pituitary development [9], a systematic, unbiased mutant screen to identify genetic regulators of CC development has not been previously reported. Here we used genetic screens and gain-of-function studies to investigate specification of CC cell lineage. From a genetic deficiency screen, we discovered that Notch signaling factors are essential regulators of CC development. Our studies demonstrate that Notch signaling controls the number of emerging CC precursor cells. We unexpectedly found that CC cells develop from head mesoderm. Expression of tinman in head mesoderm is regulated by Notch signaling and the combination of tinman and daughterless is sufficient to specify programs leading to ectopic development of CC cell precursors and their AKH+ progeny. Thus our studies reveal genetic and cellular mechanisms underlying precursor specification and expansion of neuroendocrine cells crucial for metabolic homeostasis in Drosophila. A deficiency screen identifies novel regulators of corpora cardiaca development To identify regulators of corpora cardiaca development, we screened 292 lines from the DrosDel deficiency collection [12], The requirement for glucose regulation is conserved in metazoans and crucial for metabolism, growth, and survival. In fruit flies and other insects, neurons secrete insulin-like hormones and neuroendocrine corpora cardiaca cells secrete adipokinetic hormone, a peptide with functional similarities to glucagon. Both hormones are essential for systemic glucose control in Drosophila. To understand the mechanisms governing formation and function of corpora cardiaca cells, we sought to identify their embryonic origin and investigate their developmental genetic regulation. Based on prior reports suggesting a neuroectodermal origin, we were surprised to discover using genetic lineage tracing methodsthat embryonic corpora cardiac progenitors derive from anterior head mesoderm. To our knowledge, this is the first demonstration of neuroendocrine differentiation from mesoderm in Drosophila. Genetic studies reveal that Notch signaling restricts the number of corpora cardiaca progenitors, and we show that Notch signaling inactivation results in significant expansion of corpora cardiac cells. Loss- and gain-of-function studies identified transcription factors both necessary and sufficient for corpora cardiaca development. These and other findings reveal similarities in the development of fly corpora cardiaca cells and mammalian neuroendocrine cells that develop in the pancreas, pituitary, and from neural crest. corresponding to approximately 50% of the genome. We generated strains harboring th (...truncated)