The bHLH Transcription Factor Hand Regulates the Expression of Genes Critical to Heart and Muscle Function in Drosophila melanogaster

RESEARCH ARTICLE The bHLH Transcription Factor Hand Regulates the Expression of Genes Critical to Heart and Muscle Function in Drosophila melanogaster Benjamin Hallier1, Julia Hoffmann2, Thomas Roeder2, Markus Tögel3, Heiko Meyer1, Achim Paululat1* 1 Department of Zoology/Developmental Biology, University of Osnabrück, 49069 Osnabrück, Germany, 2 Department of Animal Physiology, University of Kiel, 24098 Kiel, Germany, 3 Weatherall Institute of Molecular Medicine, University of Oxford, OX3 9DS Oxford, United Kingdom * OPEN ACCESS Citation: Hallier B, Hoffmann J, Roeder T, Tögel M, Meyer H, Paululat A (2015) The bHLH Transcription Factor Hand Regulates the Expression of Genes Critical to Heart and Muscle Function in Drosophila melanogaster. PLoS ONE 10(8): e0134204. doi:10.1371/journal.pone.0134204 Editor: Barbara Jennings, Oxford Brookes University, UNITED KINGDOM Received: November 25, 2013 Accepted: July 8, 2015 Published: August 7, 2015 Copyright: © 2015 Hallier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the German Research Foundation to A.P. (SFB 944: Physiology and dynamics of cellular microcompartments) and T.R. (Cluster of Excellence 306: Inflammation at Interfaces), by the “Incentive Award of the Faculty of Biology/Chemistry” (University of Osnabruck) to H.M., and by a grant from the FAZIT foundation to B.H. 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. Abstract Hand proteins belong to the highly conserved family of basic Helix-Loop-Helix transcription factors and are critical to distinct developmental processes, including cardiogenesis and neurogenesis in vertebrates. In Drosophila melanogaster a single orthologous hand gene is expressed with absence of the respective protein causing semilethality during early larval instars. Surviving adult animals suffer from shortened lifespan associated with a disorganized myofibrillar structure being apparent in the dorsal vessel, the wing hearts and in midgut tissue. Based on these data, the major biological significance of Hand seems to be related to muscle development, maintenance or function; however, up to now the physiological basis for Hand functionality remains elusive. Thus, the identification of genes whose expression is, directly or indirectly, regulated by Hand has considerable relevance with respect to understanding its biological functionality in flies and vertebrates. Beneficially, hand mutants are viable and exhibit affected tissues, which renders Drosophila an ideal model to investigate up- or downregulated target genes by a comparative microarray approach focusing on the respective tissues from mutant specimens. Our present work reveals for the first time that Drosophila Hand regulates the expression of numerous genes of diverse physiological relevancy, including distinct factors required for proper muscle development and function such as Zasp52 or Msp-300. These results relate Hand activity to muscle integrity and functionality and may thus be highly beneficial to the evaluation of corresponding hand phenotypes. Introduction Basic helix-loop-helix (bHLH) transcription factors are key regulators of numerous developmental processes including cardiovascular development, hematopoiesis, stem cell maintenance, PLOS ONE | DOI:10.1371/journal.pone.0134204 August 7, 2015 1 / 15 Hand Regulates the Expression of Muscle Critical Genes neurogenesis and myogenesis (reviewed by [1]). Among the class II bHLH family, Hand proteins constitute a prominent class regulating, e.g., trophoblast development, limb bud outgrowth, branchial arch development or cardiogenesis [2–14]. In higher vertebrates, e.g. mouse or human, two paralogous hand genes (hand1, hand2) are present in the genome, with both being expressed in the lateral plate mesoderm, distinct neural crest cells and the developing heart. With respect to the latter tissue, expression of both genes is initially overlapping. Upon formation of a linear heart tube however, hand1 expression becomes restricted to the developing left ventricle, while hand2 is still expressed throughout the complete heart primordium. As soon as cardiac looping initiates, hand2 expression becomes restricted to the heart region the right ventricle arises from [5, 15, 16]. Thus, the two paralogous hand genes in higher vertebrates may have evolved different tissue specific functionalities during cardiogenesis. A critical function of Hand in cardiogenesis was demonstrated by analyzing the phenotypes of loss of function mutations in mice where severe malformations of the heart were observed in animals lacking Hand activity [5, 6, 17]. For instance, mice being mutant for both, hand1 and hand2 are embryonic lethal and, among other cardiac defects, characterized by severe ventricular hypoplasia [18]. In addition, Natarahan and colleagues reported that hand1 expression is almost absent in hearts of patients suffering from ischemic or dilated cardiomyopathy, whereas hand2 expression remains unchanged [19]. In Drosophila, only a single Hand orthologue was discovered by extensive genome-wide searches for bHLH sequences [20]. Notably, Hand represents the only transcription factor identified so far that is expressed in all three major embryonic cell types that comprise the Drosophila circulatory system (cardioblasts, pericardial cells, and hematopoietic progenitors in the lymph gland). In addition to these cells, Drosophila hand is expressed in the circular visceral musculature and in distinct cells belonging to the central nervous system [21–25]. Despite its expression in the embryonic tissues mentioned above, Hand knock-out phenotypes manifest primarily in the dorsal vessel and the gut of adult animals, indicating an essential role of Hand in the remodeling of these tissues during metamorphosis rather than being required for proper determination and differentiation of the respective tissues during embryogenesis. Apparently, the phenotypes are mainly characterized by an abnormal arrangement of muscle fibers in the corresponding tissues [24]. This observation together with recent data that also describe severe disarrangements in muscle cells of hand mutant wing hearts [26] indicates that lack of Hand activity predominantly affects muscle structure or integrity. Here we took advantage of the fact that the Drosophila genome carries only a single hand orthologue and that homozygous hand mutant individuals survive into adulthood at significant rates. Cardiogenesis initially proceeds normally in such animals, however, the mature heart displays several structural malformations including sarcomere def (...truncated)