Genetic and molecular aspects of spinocerebellar ataxias

The group of spinocerebellar ataxias (SCAs) includes more than 20 subgroups based only on genetic research. The “ataxia genes” are autosomal; the “disease-alleles” are dominant, and many of them, but not all, encode a protein with an abnormally long polyglutamine domain. In DNA, this domain can be detected as an elongated CAG repeat region, which is the basis of genetic diagnostics. The polyglutamine tails often tend to aggregate and form inclusions. In some cases, protein–protein interactions are the key to understanding the disease. Protein partners of ataxia proteins include phosphatases and components of chromatin and the transcriptional machinery. To date, investigation of spinocerebellar ataxias involves population genetics, molecular methods, and studying model organisms. However, there is still no efficient therapy for patients. Here, we review the genetic and molecular data gained on spinocerebellar ataxias

Variation of NimC1 expression in Drosophila stocks and transgenic strains.

The NimC1 molecule has been described as a phagocytosis receptor, and is being used as a marker for professional phagocytes, the plasmatocytes, in Drosophila melanogaster. In studies including tumor-biology, developmental biology, and cell mediated immunity, monoclonal antibodies (P1a and P1b) to the NimC1 antigen are used. As we observed that these antibodies did not react with plasmatocytes of several strains and genetic combinations, a molecular analysis was performed on the structure of the nimC1 gene. In these strains we found 2 deletions and an insertion within the nimC1 gene, which may result in the production of a truncated NimC1 protein. The NimC1 positivity was regained by recombining the mutation with a wild-type allele or by using nimC1 mutant lines under heterozygous conditions. By means of these procedures or using the recombined stock, NimC1 can be used as a marker for phagocytic cells in the majority of the possible genetic backgrounds

Innate immunity

In this review, we discuss how studying the Drosophila immune system contributes to a better understanding of the basic principles of innate immunity. We describe the homologies between the insect and the vertebrate immune-regulatory mechanisms and convergent evolutionary traits of the Drosophila and the vertebrate immune system

There and back again: The mechanisms of differentiation and transdifferentiation in Drosophila blood cells

Transdifferentiation is a conversion of an already differentiated cell type into another cell type without the involvement of stem cells. This transition is well described in the case of vertebrate immune cells, as well as in Drosophila melanogaster, which therefore serves as a suitable model to study the process in detail. In the Drosophila larva, the latest single-cell sequencing methods enabled the clusterization of the phagocytic blood cells, the plasmatocytes, which are capable of transdifferentiation into encapsulating cells, the lamellocytes. Here we summarize the available data of the past years on the plasmatocyte-lamellocyte transition, and make an attempt to harmonize them with transcriptome-based blood cell clustering to better understand the underlying mechanisms of transdifferentiation in Drosophila, and in general

Peeling Back the Layers of Lymph Gland Structure and Regulation

During the past 60 years, the fruit fly, Drosophila melanogaster, has proven to be an excellent model to study the regulation of hematopoiesis. This is not only due to the evolutionarily conserved signalling pathways and transcription factors contributing to blood cell fate, but also to convergent evolution that led to functional similarities in distinct species. An example of convergence is the compartmentalization of blood cells, which ensures the quiescence of hematopoietic stem cells and allows for the rapid reaction of the immune system upon challenges. The lymph gland, a widely studied hematopoietic organ of the Drosophila larva, represents a microenvironment with similar features and functions to classical hematopoietic stem cell niches of vertebrates. Lymph gland studies were effectively supported by the unparalleled toolkit developed in Drosophila, which enabled the high-resolution investigation of the cellular composition and regulatory interaction networks of the lymph gland. In this review, we summarize how our understanding of lymph gland structure and hematopoietic cell-to-cell communication evolved during the past decades and compare their analogous features to those of the vertebrate hematopoietic stem cell niche

Inaktiváló hatású fehérjekomplexek szerepe és működése a Drosophila homeotikus gének szabályozásában = Function of silencing complexes in the regulation of homeotic genes in Drosophila

Csoportunk a génkifejeződés epigenetikus szabályozását tanulmányozza a Drosophila bithorax génkomplexének, mint modellrendszernek a segítségével. Az elmúlt pályázati periódusban megkezdtük egy, a csoportunk által korábban létrehozott nagyméretű mutáns gyűjteménynek a feldolgozását, amely ismeretlen, Pc- és trx csoporba tartózó gének mutációit tartalmazza. Gyűjteményünk felhasználásával jellemeztük a grappa gént, ami az élesztő DOT1 fehérjéjének, egy, a H3-as hiszton K79-es pozícióját specifikusan metiláló enzim muslica homológját kódolja. Kimutattuk, hogy a korábban kizárólag transzkripciós aktivátornak gondolt GRAINYHEAD fehérje hozzájárul ahhoz, hogy a különböző PcG fehérjekomplexek specifikusan kötődjenek az iab-7 PRE-hez). Transgenikus vonalak felhasználásával térképeztünk egy trithorax response element-et (TRE) közvetlenül az iab-7 PRE szomszédságában. Kimutattuk, hogy az ENHANCER OF ZESTE (E(Z)) fehérje C-terminális darabja histon-metil-transzferáz aktivitást mutat, és hogy egy funkciónyeréses mutáns változat, az E(Z)TRM specificitása és hatékonysága csak kis mértékben különbözik a vad fehérje megfelelő tulajdonságaitól. Genetikai interakciós vizsgálataink felvetik azt a lehetőséget, hogy az E(Z) és különböző trxG fehérjék kompetálnak azokért a nukleoszómákért, amelyek közel vannak ahhoz a helyhez (PRE és TRE), ahová ezek e fehérjék specifikusan kötődnek. Jelentős előrehaladást értünk el több, általunk azonosított, Pc- vagy trx csoportba tartozó génnek a jellemzésében is. | Our group studies the epigenetic control of gene expression using the Drosophila bithorax complex as a model system. During the past granting period we begin to characterize a large mutant collection, containing new mutations belonging to either to the Pc-, or to the trx group of genes, that we generated earlier. Using this set of mutations we described and characterized the grappa gene that codes for the sole Drosophila homolog of the yeast DOT1 protein, a histone H3 K79 methyl-transferase. Also based on our mutations, we demonstrated that the GRAINYHEAD protein, which was originally thought to be a transcriptional activator, contributes to the specific targeting of PcG protein complexes to the infraabdominal-7 Polycomb response element, iab-PRE. Using transgenic lines, we mapped a trithorax response element (TRE) next to the iab-PRE. We showed that a truncated version of the PcG protein, ENHANCER OF ZESTE (E(Z)), possess histone-methyl-transferase activity, and that the specificity of a mutant, excess of function variant, E(Z)TRM, is only slightly different from the wild type. Genetic interaction studies suggested that E(Z) and different TRXG proteins may compete for common substrates, e. g. nucleosomes near to the site where these proteins are specifically targeted. We also made significant progress in characterizing several newly identified genes belonging either to the Pc-, or to the trx

Cell lineage tracing reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments in Drosophila melanogaster

Much of our knowledge on hematopoiesis, hematopoietic compartments, hematopoietic cell lineages and immunity has been derived from studies on the vertebrate immune system. The sophisticated innate immunity of insects, the phylogenetic conservation and the power of Drosophila genetics allowed the investigation of immune cell (hemocyte) lineage relationships in Drosophila melanogaster. The development of the hemocyte lineages in Drosophila is a result of a precisely regulated succession of intracellular and intercellular events, though the nature and extent of these interactions are not known. We describe here a cell lineage tracing system set up to analyze the development of hemocyte lineages and functionally distinct hemocyte subsets. This system allowed us to distinguish two major embryonic hemocyte lineages, the crq and Dot lineages, in two, physically separated compartments, the embryonic macrophages and the embryonic lymph gland. We followed the fate and development of these lineages in the construction of the larval hematopoietic compartments and during the cell-mediated immune response, the encapsulation reaction. Our results revealed the considerable plasticity and concerted action of the hematopoietic compartments and the hemocyte lineages in the development of the innate immune system and in the course of the cell-mediated immune response in Drosophila

Multi-Dimensional Immuno-Profiling of Drosophila Hemocytes by Single Cell Mass Cytometry

Single cell mass cytometry (SCMC) combines features of traditional flow cytometry (FACS) with mass spectrometry and allows the measurement of several parameters at the single cell level, thus permitting a complex analysis of biological regulatory mechanisms. We optimized this platform to analyze the cellular elements, the hemocytes, of the Drosophila innate immune system. We have metal-conjugated six antibodies against cell surface antigens (H2, H3, H18, L1, L4, P1), against two intracellular antigens (3A5, L2) and one anti-IgM for the detection of L6 surface antigen, as well as one anti-GFP for the detection of crystal cells in the immune induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive, in immune induced states, in tumorous mutants (hopTum bearing a driver mutation and l(3)mbn1 carrying deficiency of a tumor suppressor) as well as in stem cell maintenance defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets, lamellocytes, plasmatocytes, crystal cell, and delineated the unique immunophenotype of the mutants. We have identified sub-populations of L2+/P1+ (l(3)mbn1), L2+/L4+/P1+ (hopTum) transitional phenotype cells in the tumorous strains and a sub-population of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time, that mass cytometry, a recent single cell technology combined with multidimensional bioinformatic analysis represents a versatile and powerful tool to deeply analyze at protein level the regulation of cell mediated immunity of Drosophila

A Drosophila melanogaster sejt-közvetítette immunitása = The cell mediated immunity of Drosophila melanogaster

A Projekt keretében a.) a vérsejtmarker panelt in vivo lamellocita markerrel egészítettük ki. b.) a Trol molekulát, az apoptotikus sejteket felismerő receptorként definiáltuk. c.) jellemeztük az immunkompartmentumok funkcióit és meghatároztuk a sejtes elemek eredetét. A vérsejtképző kompartmentumok az embrióban és a lárvában egymástól elhatároltak, míg az immunválasz során valamennyi kompartmentum részt vesz a vérsejtek képzésében. A lamellociták fejlődése több lépésben zajlik a szesszilis szövet kezdeti és fő részvételével. A lamellociták plazmatocita jellegű sejtekből képződnek ezért igazoltnak látjuk, hogy a fagocita sejtek nem terminálisan differenciálódott sejtek, hanem immunindukciót követően lamellocitákká alakulhatnak, mely nagyfokú plaszticitásukat mutatja. d.) immun-modulként jellemezhető génklasztert azonosítottunk Drosophilában és egyéb rovarfajokban. A génklaszter egyes tagjai részt vesznek a mikróbák opszonizálásában, sejthez kötésében és a fagocitózis folyamatában. e.) a sejt közvetítette immunitás eddig ismeretlen formáját találtuk és jellemeztük egyes Drosophila fajokban. A tokképző reakcióban sokmagvú óriássejtek vesznek részt, melyek a gerinces szervezetek egyes granulómáira jellemző óriássejtekhez hasonlítanak. f.) veleszületett immunitás témájú kurzusokat tartottunk és szakdolgozókat valamint Ph.D. hallgatókat foglalkoztatunk a laboratóriumban. | In the framework of the Project we: a.) expanded the hemocyte marker panel with an in vivo lamellocyte marker. b.) defined Trol as a receptor molecule for apoptotic cells on plasmatocytes. c.) characterized the cellular elements of the immune compartments with respect to function and origin. The hematopoietic compartments are separated during the embryonic and larval development however, in the course of the cell mediated immune response all compartments contribute to the development of effector cells. The differentiation of lamellocytes occurs in sequence with the initial and major contribution of the sessile hematopoietic tissue. The lamellocytes differentiate from precursors with the characteristics of plasmatocytes therefore it is proposed that phagocytic cells are not terminally differentiated cells but upon immune stimulation they may become lamellocytes, illustrating their striking plasticity. d.) identified a gene cluster instrumental in cell mediated immunity in Drosophila and in insects, in general. The members of the gene cluster encode proteins apparently involved in binding, opsonization, and phagocytosis of bacteria. e.) found a novel form of the cell mediated immunity of Drosophila species. The encapsulation reaction involves the contribution of multinucleated giant hemocytes resembling to multinuclear giant cells in granuloma formation in vertebrates. f.) ran courses in innate immunity and trained undergraduate and graduate students in the laboratory