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Research report:
AG Renkawitz-Pohl


Renkawitz-Pohl
Renate Renkawitz-Pohl

Diploma (Biology), University of Düsseldorf, 1974
Dr. rer. nat. (Genetics), University of Düsseldorf, 1977
Postdoc, Brown University, Providence, USA, 1977-1978
Postdoc (Genetics), University of Düsseldorf, 1979-1980
Postdoc (Molecular Genetics), University of Heidelberg, 1981-1984
Habilitation (Biology), University of Heidelberg, 1985
Group Leader, Genzentrum, Max-Planck-Institut für Biochemie Martinsried, 1985-1991
Professor (C3) for Genetics, Philipps-University, Marburg 1991-1995
Professor (C4) for Developmental Biology, Philipps-University, Marburg 1996-present





Developmental Biology
projects:






Developmental Biology


Our research interest focuses mainly on the development of the body wall and gut musculature, the development of the heart, and on regulatory processes during spermatogenesis. Most analyses are performed with the genetically and developmentally well accessibly organism Drosophila melanogaster, but we have extended our studies to vertebrate cell culture systems. We apply a wide range of methods to functionally analyse these developmental processes (immunhistology, recombinant DNA technology, molecular and classical genetics, protein-protein interaction studies, electronmicroscopy).



Rolling pebbles and the formation of the body wall musculature

In Drosophila, the body wall musculature of the larvae consists of approximately 30 distinct muscles per hemisegment. This muscle pattern is prefigured by 30 founder cells which express the IgSF member Dumbfounded (Duf) and Roughest (Rst). These cells recruit fusion competent myoblasts (fcms) to form syncytia of 4 - 24 nuclei. Sticks and stones (Sns) - another IgSF member, is expressed in fusion competent myoblasts and essential for heterologous cell adhesion (for reviews see Chen and Olson, 2004; Abmayr et al., 2005). Our main interest is to gain further insight into myoblast fusion. We previously found that Rolling pebbles (Rols) is expressed during fusion in founder/precursor cells, and is essential for the second wave of fusion. Rolling pebbles is a cytoplasmic multidomain protein with ring finger ankyrin repeats and TPR repeats (Rau et al., Development 128, 5061-5073, 2001; reviewed in Holz et al., 2004). We established antibodies against Rols. These allowed us to show that Rols forms an adhesive ring together with Duf on the site of the growing muscle, while Sns, another IgSF member, forms the complementary structure on the site of the fusion competent myoblast (Kesper, Dissertation 2005; Kesper et al., manuscript in preparation). We found that Rols is a component of the Z-Discs of the sarcomeres where Rols colocalises and likely interacts with Alpha-Actinin and the Sls isoform Zormin. Thus we proposed that Rols links these molecules in the Z-discs. We showed furthermore that Rolling pebbles, Dumbfounded/Kirre, Alpha-Actinin and Zormin are components of the terminal Z-discs which link the muscle to the epidermal attachment sites. In yeast-two-hybrid assays, direct molecular interactions between Rolling pebbles and the colocalized proteins were shown. In comparison to known in vivo properties of Rolling pebbles domains we propose a model for the relevance of the individual Rols domains and their relevance for myoblast fusion, sarcomer asssembly and stability as well as attachment formation and maintainance (Reichert, Diplomarbeit 2004; Kreisköther Dissertation 2005; Kreisköther et al., 2006 in press). Our previous analysis of the rolling pebbles transcription unit revealed two promoters leading to a rols7 and a rols6 transcript. We created a specific rols6 loss-of-function mutant, which showed defects in the morphogenesis of Malphigian tubules while the musclulature develops properly. This shows that rols7 is the myogenesis-relevant transcript (Pütz, Dissertation 2004; Pütz et al., 2005). The distinct tissue-specific promoter and enhancer elements of rols have been determined (Pütz, Dissertation, 2004; Kesper, Dissertation, 2005, Kesper, Pütz et al., in Vorbereitung).



Identification of new components of the myoblast fusion process

We screened a collection of EMS-induced mutations from Prof. Klämbt’s laboratory, Münster, with a mesoderm-specific marker for defects in myoblast fusion. In bubbles mutants, myoblast fusion stops at the mono-nuclear founder cell stage (Reichert, Diplomarbeit, 2004). Also in schizo mutants myoblast fusion stops at this step. Schizo encodes an ARF6 nucleotide exchange factor, which has been previously identified by its role in nervous system development (Önel et al., 2004). Whereas in other mutants like for example schwächling, (Berger, Diplomarbeit, 2005) muscle precursor cells with three to four nuclei are formed but then they do not proceed to the second wave of fusion which leads to the final size of synzytia. Also in kette mutants, fusion arrests after precursor formation. Kette encodes the HEM homolog of Drosophila and is a regulator of F-Actin organisation in the nervous system (Bogdan and Klämbt, 2003). Epistasis experiments show that Kette genetically interacts with Blow during myoblast fusion. We propose that Blow and Kette successfully signal cell adhesion during myoblast fusion (Schröter et al., Development 2004). We started to analyse the signaling properties of Blow (Sickmann, Diplomarbeit, 2005). With the identification of a WASp mutant with fusion defects we identified a further component which is relevant for myoblast fusion (Schäfer, Diplomarbeit, 2005). For bubbles and schwächling we started to map the chromosomal position and identification of the responsible gene by testing the chromosome carrying the EMS mutant against deficiencies (Berger, Diplomarbeit, 2005; Wierach, Diplomarbeit, 2005).



Development of the circular visceral musculature

The visceral musculature forms two layers around the gut. Determination of founder cells in the visceral mesoderm depends on Jeb/Alk signaling (Stute, Dissertation 2004; Stute et al., Development 131, 743-754, 2004). The circular musculature arises by fusion of one DUF and one SNS expressing cell, the cells flatten and span one hemisegment. Thereafter, founder cells for the longitudinal visceral muscles migrate over the circular muscles and fuse with the remaining SNS-positive cells along the trunk mesoderm (Klapper et al., 2002, Mech. Dev. 110, 85-96). At the ultrastructural level, we observed no prefusion complexes or electrondense plaques. This argues for a fusion mechanism that is distinct from the second fusion wave in the somatic mesoderm. The circular visceral muscles are web-shaped and the longitudial musculature is interwoven to form a net around the gut as revealed by raster EM. Stretching and shaping of the circular visceral muscles depends on Blow and Kette while syncytia formation is independent of these proteins (Schröter et al., 2006, Differentiation in revision).



Drosophila spermatogenesis and chromatin remodeling

Spermatogenesis is a highly conserved biological process. Stem cells go through asymmetric divisions giving rise to one stem cell and one spermatogonium. Spermatogonia go through a distinct number of mitotic amplification steps and then enter the extended meiotic prophase with high transcriptional activity. After meiosis and during spermiogenesis the needle-shaped head is formed and the sperm tail is assembled. Many mRNAs which encode proteins for spermiogenesis are synthesized during the meiotic prophase, but translationally repressed until they are required during sperm morphogenesis. As in mammals, the male genome is highly condensed in the mature sperm. In mammals, first transition proteins and then protamines replace the majority of histones in the chromatin (for a review on spermatogenesis see Renkawitz-Pohl et al., 2005). don juan and don juan-like encode proteins of the spermatide nucleus and the flagellum. Don Juan, Don Juan-like and Min might be candidates for transition proteins (Hempel, Dissertation, 2004; Hempel et al., Dev. Dyn. 2006, in press). Besides two protamines we recently identified Mst77 as a further specific component of the sperm chromatin, in which histones are hardly detectable. The switch between the histone-based nucleosomal configuration of the chromatin and the protamine/Mst77-based configuration takes place in spermatides when the nucleus is canoe-shaped (Jayaramaiah Raja, Dissertation 2005; Jayaramaiah Raja and Renkawitz-Pohl 2005). Currently our research focuses on the „Why“ and „How“ of chromatin reorganisation during sperm formation. All these genes, which encode chromosomal proteins, are transcribed during meiotic prophaseI in dependence on the tissue-specific TAFII80 Cannonball. Translation is repressed for several days until mid spermiogenesis, this is achieved by distinct elements in the 5´UTR of don juan, don juan like, min, protamineA, protamineB and Mst77 mRNAs (Hempel, Dissertation 2004; Jayaramaiah, Dissertation 2005; Barckmann, Diplomarbeit 2005; Leidert, Diplomarbeit 2005; Jayaramaiah Raja and Renkawitz-Pohl 2005; Hempel et al. 2006, Dev. Dyn. in press).



Publications

Stute, C., Schimmelpfeng, K., Renkawitz-Pohl, R., Palmer, R. H. and Holz, A. (2004). Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways (miliAlk) as receptor for Jeb signalling. Development 131, 743-754.

Schröter, R., Lier, S., Holz, A., Bogdan, S., Klämbt, C., Beck, L. and Renkawitz-Pohl, R. (2004). kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila. Development 131, 4501-4509.

Jayaramaiah Raja, S. and Renkawitz-Pohl, R. (2005). Replacement by Drosophila melanogaster protamines and Mst77F of histones during chromatin condensation in late spermatids and role of Sesame in the removal of these proteins from the male pronucleus. Mol. Cell. Biol. 25, 6165-6177.

Moon, H., Filippova, G., Loukinov, D., Pugacheva, E., Chen, Q., Smith, S.T., Munhall, A., Grewe, B., Bartkuhn, M., Arnold, R., Burke, L. J., Renkawitz-Pohl, R., Ohlsson, R. Zhou, J. Renkawitz, R. and Victor Lobanenkov, V. (2005). CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator. EMBO Rep. 6, 165-170.

Pütz, M., Kesper, D. A., Buttgereit, D. and Renkawitz-Pohl, R. (2005). In Drosophila melanogaster, the Rolling Pebbles isoform 6 (Rols6) is essential für proper Malpighian tubule morphology. Mech. Dev. 122, 1206-1217.

Reviews


Holz, A., Buttgereit, D. and Renkawitz-Pohl, R. (2004). Bildung der Muskulatur im Drosophila Embryo: Funktion von Rolling pebbles. BIOspektrum 1/04
Renkawitz-Pohl, R., Hollmann, M., Hempel, L. and Schäfer, M.A. (2005). Spermatogenesis. In: Comprehensive Molecular Insect Science, Vol. 1: Reproduction and Development. Elsevier BV. Editors: Lawrence I. Gilbert, Kostas Iatrou, Sarjeet S. Gill, pp. 157-177.

Pühler et. al including Buttgereit, D. and Renkawitz-Pohl, R. (2005). Biotechnology, eds. H. J. Rehm and G. Reed "Genomics and Bioinformatics, Volume editor C. W. Sensen", Wiley-VHC Chapter 1: Genom Projects of Model Organisms, Volume , actualized version in press


Diploma theses

Klim, Manuela (2004): Phänotypische Charakterisierung der Myogenese in Embryonen mit Mutationen im klötzchen-Gen in Drosophila melanogaster

Müller, Susanne (2004): Phänotypische Charakterisierung und genetische Kartierung von Mutationen in Myoblasten-Fusions-relevanten Genen auf dem 3. Chromosom von Drosophila melanogaster

Reichert, Nina (2004): Entwicklung von Myotuben bei Drosophila melanogaster: Analyse zweier EMS induzierter Mutanten und Protein-Protein-Wechselwirkungen zwischen Rolling pebbles und weiteren Fusions-relevanten Proteinen

Sarges, Jan Phillip (2004): Bedeutung der DOCK-Proteinfamilie in wandernden Muskelvorläuferzellen der Maus (extern: Prof. Dr. Carmen Birchmeier, Max-Delbrück-Zentrum, Berlin)

Schäfer, Gritt (2005): Molekulare Charakterisierung und funktionelle Analyse zweier Gene, die für die Myoblastenfusion des Drosophila Embryos essentiell sind

Barckmann, Bridlin (2005): Analyse der Transkriptions- und Tanslationskontrolle zur Synthese des Mst77F-Proteins, einer Komponenten des kondensierten Chromatins des Spermiums in Drosophila melanogaster

Sickmann, Angela (2005): Blow wird durch Phosphorylierung post-translational modifiziert und interagiert nicht mit der Spleißvariante B des Adaptorproteins Crk

Leidert, Eva (2005): Die testisspezifisch exprimierten Gene min (CG 1988) und CG 31861 besitzen äußerst kurze Promotorbereiche und ihre mRNA unterliegt einer Translationskontrolle

Wang, Shuoshuo (2005): Charakterisierung von Mutanten einer membranintegralen Protease Kuzbanian bei Drosophila melanogaster

Berger, Susanne (2005): Analyse der Protein-Interaktionen von Rolling pebbles in vivo und Identifizierung eines in der zweiten Stufe der Myoblastenfusion beteiligten Gens von Drosophila melanogaster

Wierach, Barbara (2005): Charakterisierung von bubbles, einem Gen für Zelltypspezifizierung in der Myogenese von Drosophila melanogaster und die Analyse von Protein-Interaktionen von Rolling pebbles unter Anwendung des CytoTrap-Systems


Doctoral theses

Kreß, Julia Anne (2004): Zelluläre Funktionen der Hyaluronsäure-bindenden Proteinase (HABP): Aufklärung intrazellulärer Signalwege in humanen Endothelzellen (extern: PD Dr. Johannes Dodt, Paul-Ehrlich-Institut, Langen)

Hahn, Silvia (2004): Das endogene Retrovirus HTDV/HERV-K: Untersuchungen zur Funktion des akzessorischen Proteins Rec (extern: Prof. Dr. Johannes Löwer, Paul-Ehrlich-Institut, Langen)

Hempel, Leonie (2004): Kurze Promotoren und Translationskontrollelemente steuern die Expression von Don Juan, Don Juan-Like und Min, wobei Don Juan und Don Juan-Like vermutlich funktionell redundant in der Spermiogenese von Drosophila melanogaster sind

Pütz, Michael (2004): Genregulatorische und funktionelle Analysen des differentiell exprimierten rolling pebbles Transkriptes rols6 zeigen, dass das Rols6 Protein für die vollständige Differenzierung der Malpighigefäße in Drosophila melanogaster notwendig ist

Stute, Christiana (2004): Die Determination der Founder- und fusionskompetenten Zellen des viszeralen Mesoderms von Drosophila melanogaster ist abhängig von Notch sowie Jeb/Alk vermitteltem RTK-Signalweg

Grigore, Rischitor (2005): Transcription factor Sp3 as target for SUMOylation in vivo (extern: Prof. Dr. Suske, IMT)

Paparidis, Zissis (2005): Cis-acting elements controlling the expression of the human Gli3 gene (extern: Prof. Dr. Grzeschik, Humangenetik)

Jayaramaiah Raja, Sunil (2005): Chromatin condensation during Drosophila spermiogenesis and decondensation after fertilization

Rau, Marlene (2005): Zebrafish Trap230/Med12 is required for Sox9 activity and limb (extern: Dr. Neumann, EMBL, Heidelberg)

Kesper, Dörthe Andrea (2005): Die Analyse der cis-Regulation des rols-Gens und die Beteiligung von Rols7 am Aufbau eines podosomenähnlichen adhesiven Komplexes (PILMAC), der eine zentrale Rolle in der Myoblastenfusion bei Drosophila melanogaster einnimmt

Kreisköther, Nina (2005): In der Myogenese von Drosophila melanogaster interagiert Rolling pebbles 7 in den Z-Scheiben der Sarkomere mit alpha-Aktinin und D-Titin/Kettin/Zormin, in der terminalen Z-Scheibe kolokalisiert es zudem mit Dumbfounded/Kirre


Structure of the group (12/2005)

Group leader

Prof. Dr. Renate Renkawitz-Pohl

Secretary

Heike Sauer

Academic staff

Dr. Detlev Buttgereit
Dr. Susanne Önel

Postdoctoral fellows

Dr. Sunil Jayaramaiah Raja
Dr. Christiana Stute

PhD students

Dörthe Kesper
Nina Kreisköther
Christina Rathke
Roxane Schröter
Gritt Schäfer
Angela Sickmann
Susanne Berger
Bridlin Barckmann
Barbara Wierach

Diploma/L3 students

Stefan Awe
Bettina Bonn
Christine Dottermusch
Aurelia Fuchs
Matthias Jacobs
Tanja Klein
Lisa Wirth

Technical assistants

Nadine Müller
Ruth Hyland
Sabina Huhn


Grants

Renkawitz-Pohl: Internationales Graduiertenkolleg 767 „Transcriptional Control in Developmental Processes“, Förderzeitraum: 01.01.2002 bis 31.12.2004

Sachbeihilfe der DFG - RE 628/12-1: „Chromatin in sperm“, bewilligt am 17.02.04 für 2 Jahre

Sachbeihilfe der DFG - RE 628/12-2: „Chromatin in sperm“, bewilligt am 28.11.2005 für 1 Jahr

Sachbeihilfe der DFG - RE 628/14-2: „Myogenese“, bewilligt am 18.04.2005 für 3 Jahre

EU Projekt Network of Excellence, Myores, Förderzeitraum 01.01.2005 bis 31.12.2009


Address

Prof. Dr. Renate Renkawitz-Pohl
Entwicklungsbiologie
Fachbereich Biologie
Philipps-Universität
Karl-von-Frisch-Str. 8
35043 Marburg

Tel: +49 6421 28-21503
Fax: +49 6421 28-21538
renkawit@staff.uni-marburg.de

Zuletzt aktualisiert: 18.05.2006 · dohle

 
 
 
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