0000000000235515
AUTHOR
Benjamin Altenhein
Structures of two molluscan hemocyanin genes: significance for gene evolution.
We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini , were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis , corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exh…
Expression profiling of glial genes during Drosophila embryogenesis
AbstractIn the central nervous system of Drosophila, the induction of the glial cell fate is dependent on the transcription factor glial cells missing (gcm). Though a considerable number of other genes have been shown to be expressed in all or in subsets of glial cells, the course of glial cell differentiation and subtype specification is only poorly understood. This prompted us to design a whole genome microarray approach comparing gcm gain-of-function and, for the first time, gcm loss-of-function genetics to wildtype in time course experiments along embryogenesis. The microarray data were analyzed with special emphasis on the temporal profile of differential regulation. A comparison of bo…
The Sequence of a Gastropod Hemocyanin (HtH1 from Haliotis tuberculata)
The eight functional units (FUs), a-h, of the hemocyanin isoform HtH1 from Haliotis tuberculata (Prosobranchia, Archaeogastropoda) have been sequenced via cDNA, which provides the first complete primary structure of a gastropod hemocyanin subunit. With 3404 amino acids (392 kDa) it is the largest polypeptide sequence ever obtained for a respiratory protein. The cDNA comprises 10,758 base pairs and includes the coding regions for a short signal peptide, the eight different functional units, a 3'-untranslated region of 478 base pairs, and a poly(A) tail. The predicted protein contains 13 potential sites for N-linked carbohydrates (one for HtH1-a, none for HtH1-c, and two each for the other si…
Identity, origin, and migration of peripheral glial cells in the Drosophila embryo.
Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. Here we studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows…
Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression
In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines we have clarified the lineages of the lateral glia in the embryonic ventral nerve cord and linked each glial cell to a specific stem cell. For the lineage of the longitudinal glioblast we show that it consists of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm targ…
Myelin Basic Protein synthesis is regulated by small non‐coding RNA 715
Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein.
Abalone (Haliotis tuberculata) hemocyanin type 1 (HtH1) . Organization of the = 400 kDa subunit, and amino acid sequence of its functional units f, g and h
We have identified two separate hemocyanin types (HtH1 and HtH2) in the European abalone Haliotis tuberculata. HtH1/HtH2 hybrid molecules were not found. By selective dissociation of HtH2 we isolated HtH1 which, as revealed by electron microscopy and SDS/PAGE, is present as didecamers of a approximately 400 kDa subunit. Immunologically, HtH1 and HtH2 correspond to keyhole limpet hemocyanin (KLH)1 and KLH2, respectively, the two well-studied hemocyanin types of the closely related marine gastropod Megathura crenulata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, SDS/PAGE and N-terminal sequencing, we identified eight different 40-60 kDa functional unit…
Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system
International audience; One of the numerous functions of glial cells in Drosophila is the ensheathment of neurons to isolate them from the potassium-rich haemolymph, thereby establishing the blood-brain barrier. Peripheral nerves of flies are surrounded by three distinct glial cell types. Although all embryonic peripheral glia (ePG) have been identified on a single-cell level, their contribution to the three glial sheaths is not known. We used the Flybow system to label and identify each individual ePG in the living embryo and followed them into third instar larva. We demonstrate that all ePG persist until the end of larval development and some even to adulthood. We uncover the origin of al…
Subunit organization of the abalone Haliotis tuberculata hemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h.
We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abalone Haliotis tuberculata. On the basis of limited proteolytic cleavage, two-dimensional immunoelectrophoresis, PAGE, N-terminal protein sequencing and cDNA sequencing, we have identified eight different 40-60-kDa functional units (FUs) in HtH2, termed HtH2-a to HtH2-h, and determined their linear arrangement within the elongated 400-kDa subunit. From a Haliotis cDNA library, we have isolated and sequenced a cDNA clone which encodes the five C-terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg from Octo…
Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and htH.
Keyhole limpet hemocyanin (KLH) is a well-established immune stimulant and hapten carrier, and Haliotis tuberculata hemocyanin (HtH) is a related product. Biologically, KLH and HtH are blue copper proteins which serve as oxygen carriers in the blood of the keyhole limpet Megathura crenulata and the abalone H. tuberculata, respectively, two marine gastropods. Both hemocyanins occur as two distinct isoforms, termed KLH1 KLH2, HtH1, and HtH2. Each of these molecules is based on a very large polypeptide chain, the subunit (molecular mass ca 400 kDa), which is folded into a series of eight globular functional units (molecular mass ca 50 kDa each). Twenty copies of this subunit form a cylindrical…
Tyramine Actions on Drosophila Flight Behavior Are Affected by a Glial Dehydrogenase/Reductase
The biogenic amines octopamine (OA) and tyramine (TA) modulate insect motor behavior in an antagonistic manner. OA generally enhances locomotor behaviors such as Drosophila larval crawling and flight, whereas TA decreases locomotor activity. However, the mechanisms and cellular targets of TA modulation of locomotor activity are incompletely understood. This study combines immunocytochemistry, genetics and flight behavioral assays in the Drosophila model system to test the role of a candidate enzyme for TA catabolism, named Nazgul (Naz), in flight motor behavioral control. We hypothesize that the dehydrogenase/reductase Naz represents a critical step in TA catabolism. Immunocytochemistry rev…
The developmental proteome of Drosophila melanogaster
Drosophila melanogaster is a widely used genetic model organism in developmental biology. While this model organism has been intensively studied at the RNA level, a comprehensive proteomic study covering the complete life cycle is still missing. Here, we apply label-free quantitative proteomics to explore proteome remodeling across Drosophila’s life cycle, resulting in 7952 proteins, and provide a high temporal-resolved embryogenesis proteome of 5458 proteins. Our proteome data enabled us to monitor isoform-specific expression of 34 genes during development, to identify the pseudogene Cyp9f3Ψ as a protein-coding gene, and to obtain evidence of 268 small proteins. Moreover, the comparison wi…
Tracing cells throughout development: insights into single glial cell differentiation
In the article “Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system” we combined our expertise to identify glial cells of the embryonic peripheral nervous system on a single cell resolution with the possibility to genetically label cells using Flybow. We show that all 12 embryonic peripheral glial cells (ePG) per abdominal hemisegment persist into larval (and even adult) stages and differentially contribute to the three distinct glial layers surrounding peripheral nerves. Repetitive labelings of the same cell further revealed that layer affiliation, morphological expansion, and control of proliferation are predetermined and subject…
Netrins guide migration of distinct glial cells in the Drosophila embryo
Development of the nervous system and establishment of complex neuronal networks require the concerted activity of different signalling events and guidance cues, which include Netrins and their receptors. In Drosophila, two Netrins are expressed during embryogenesis by cells of the ventral midline and serve as attractant or repellent cues for navigating axons. We asked whether glial cells, which are also motile, are guided by similar cues to axons, and analysed the influence of Netrins and their receptors on glial cell migration during embryonic development. We show that in Netrin mutants, two distinct populations of glial cells are affected: longitudinal glia (LG) fail to migrate medially …
Terminal tendon cell differentiation requires the glide/gcm complex.
International audience; Locomotion relies on stable attachment of muscle fibres to their target sites, a process that allows for muscle contraction to generate movement. Here, we show that glide/gcm and glide2/gcm2, the fly glial cell determinants, are expressed in a subpopulation of embryonic tendon cells and required for their terminal differentiation. By using loss-of-function approaches, we show that in the absence of both genes, muscle attachment to tendon cells is altered, even though the molecular cascade induced by stripe, the tendon cell determinant, is normal. Moreover, we show that glide/gcm activates a new tendon cell gene independently of stripe. Finally, we show that segment p…
Notch and Numb are required for normal migration of peripheral glia in Drosophila
Abstract A prominent feature of glial cells is their ability to migrate along axons to finally wrap and insulate them. In the embryonic Drosophila PNS, most glial cells are born in the CNS and have to migrate to reach their final destinations. To understand how migration of the peripheral glia is regulated, we have conducted a genetic screen looking for mutants that disrupt the normal glial pattern. Here we present an analysis of two of these mutants: Notch and numb. Complete loss of Notch function leads to an increase in the number of glial cells. Embryos hemizygous for the weak NotchB-8X allele display an irregular migration phenotype and mutant glial cells show an increased formation of …
The transmembrane receptor Uncoordinated5 (Unc5) is essential for heart lumen formation in Drosophila melanogaster
AbstractTransport of liquids or gases in biological tubes is fundamental for many physiological processes. Our knowledge on how tubular organs are formed during organogenesis and tissue remodeling has increased dramatically during the last decade. Studies on different animal systems have helped to unravel some of the molecular mechanisms underlying tubulogenesis. Tube architecture varies dramatically in different organs and different species, ranging from tubes formed by several cells constituting the cross section, tubes formed by single cells wrapping an internal luminal space or tubes that are formed within a cell. Some tubes display branching whereas others remain linear without interse…
Gene structure and hemocyanin isoform HtH2 from the mollusc Haliotis tuberculata indicate early and late intron hot spots.
Abstract We have cloned and sequenced cDNAs coding for the complete primary structure of HtH2, the second hemocyanin isoform of the marine gastropod Haliotis tuberculata. The deduced protein sequence comprises 3399 amino acids, corresponding to a molecular mass of 392 kDa. It shares only 66% of structural identity with the previously analysed first isoform HtH1, and according to a molecular clock, the two isoforms of Haliotis hemocyanin separated ca. 320 million years ago. By genomic polymerase chain reaction and 5′ race, we have also sequenced the complete gene of HtH2 (18,598 bp), except of the 5′ region in front of the secreted protein. It encompasses 15 exons and 14 introns and shows se…