Research report:
AG 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:
- Rolling pebbles and the formation of the body wall musculature
- Identification of new components of the myoblast fusion process
- Development of the circular visceral musculature
- Drosophila spermatogenesis and chromatin remodeling
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
