Mechanisms of membrane recognition and of building a brain: Drosophila melanogaster as model system for developmental neurobiology
Team 1993-1996: Brigitte Bader*, Margit Böhler*, Liliana Falla-Christ *, Eckhart Eule*, Renate Gafert, Dieter Göhlmann, Peter Robin Hiesinger*, Beate Lichte, Johannes Korn*, Danielle Million, Zhiping Nie*, Alexandra Ochsner*, Christian Reiter*, Thorsten Schimansky, Thilo Schneider, Angela Straube*, Annette Sulzbacher, Martin Strünkelnberg*, Florian Wagner
* still lab members at the end of 1996
Guest scientists: Tanya Meyer (San Diego), Dr. Venugopala Reddy Gonehal (Bombay), Prof. Dr. Ross Cagan (St. Louis)
We use the small fly Drosophila melanogaster as a model system for developmental biology. Our main interest is an understanding of the in vivo functions of cell adhesion molecules.
Neural Recognition in Drosophila: Restricted Expression of the IrreC-rst Protein is Required for Normal Axonal Projections of Columnar Visual Neurons
Thilo Schneider, Christian Reiter, Eckhart Eule, Brigitte Bader, Beate Lichte, Zhiping Nie, Thorsten Schimansky
The 104 kDa IrreC-rst protein, a member of the immunoglobulin superfamily, mediates homophilic adhesion in cell cultures. In larval optic chiasms, the protein is found on recently formed axon bundles, not on older ones. In developing visual neuropils it is present in all columnar domains of specific layers. The number of IrreC-rst positive neuropil stratifications increases until the midpupal stage. Immunoreactivity fades thereafter. The functional importance of the restricted expression pattern is demonstrated by the severe projection errors of axons in the first and second optic chiasms in loss of function mutants and in transformants that express the IrreC-rst protein globally. Epigenesis of the phenotypes can partially be explained on the bases of homophilic IrreC-rst interactions.
The originals of the confocal images in our corresponding publication (Schneider et al., 1995) are available in the FLYBRAIN database under the accession number AB00001.
Reorganization of membrane contacts prior to apoptosis in the Drosophila retina: The role of the IrreC-rst protein
Christian Reiter, Thorsten Schimansky, Zhiping Nie
The final step of pattern formation in the developing retina of Drosophila is the elimination of supernumerary cells between ommatidia and the differentiation of the remaining cells into secondary and tertiary pigment cells. Temporally and spatially highly regulated expression of the IrreC-rst protein is essential for correct sorting of cell-cell contacts in the pupal retina without which the ensuing wave of apoptosis does not occur. IrreC-rst accumulates strongly at the borders between primary pigment and interommatidial cells. Mutant and experimental misexpression analysis show that the accumulation of the IrreC-rst protein is necessary for aligning interommatidial cells in a single row. This reorganization is a prerequisite for the identification of death candidates. The influence of IrreC-rst on retinal development demonstrates the importance of specific cell contacts for the assignment of the apoptotic fate (Reiter et al., 1996). Furthermore, we could show that misexpression of the full length IrreC-rst protein, but not the D1-construct (see below), in cone cells is able to inhibit cell death of interommatidial cells.
Studies on the in vivo function of extracellularly truncated IrreC-rst proteins
Zhiping Nie, Christian Reiter, Florian Wagner, Johannes Korn, Tanya Meyer
We have produced transformants that carry truncated versions of the irreC-rst HB3-cDNA behind five UAS-sequences. After crosses to Gal4-enhancer trap lines the F1-generation expresses extracellularly truncated versions of the IrreC-rst protein. The D1-construct is a deletion of Ig-domain 1, D1-2 of Ig-domains 1 + 2, D1-3 of Ig-domains 1 to 3, D1-4 of Ig-domains 1 to 4. Their targeted expression in the nervous system of Drosophila is compared to the effects of targeted misexpression of the full-length version of HB3 (see e.g. Schneider et al. 1995). We did not detect any remaining in vivo function of D1-3 and D1-4. When their expression was driven by neuron-specific or -non-specific Gal4-lines in the visual system (e.g. in cone cells) no mutant phenotypes could be detected.
Expression of D1 in the same lines still produced some of the phenotypes known from targeted misexpression of IrreC-rst. These defects were weaker as those known from the misexpression of the wild type protein. There is, however, one very important exception. Expression of D1 by Gal4-lines, that direct misexpression to cone cells, leads consistently to a dramatic enhancement of a lamina defect that is only weakly indicated with IrreC-rst. The extension of rhabdomeres of the retinula cells no longer stops at the base of the developing compound eye, they extent instead beyond the basement membrane, hereby displacing the lamina significantly. The Gal4-line specificity of this defect suggests that misexpression in cone cells maybe important. This is an intriguing conclusion as it is known that the feet of the cone cells normally close around the basal tips of the rhabdomeres.
Our results indicate that IrreC-rst must be a multifunctional protein with several competing interactions.
Biochemical and functional characterization of the IrreC-rst protein
For this work cultured S2 cells were transfected with cDNAs coding for full length or truncated IrreC-rst. Full lenght IrreC-rst protein is immunoreactive with anti-tyrosine phosphate antibodies, the extracellular domain alone is not. Digestion with n-glycosidase reduces the molecular weight. IrreC-rst is a homophilic cell adhesion molecule. S2-cell clusters of transfected cells readily form after heat shock. Elimination of extracellullar Ig-domains reduce the strength of homophilic interaction. However, even elimination of the four outer Ig-domains does not eliminate homophilic adhesiveness of the fifth completely. In the supernatant of transfected cells and in the haemolymph of IrreC-rst overexpressing L3 larvae a shortened and soluble isoform of IrreC-rst was detected. The molecular weight of this isoform corresponds to that of the mature extracellular domain alone. We postulate a proteolytic cleavage site near the transmembrane domain. Western blots of native PAGE gels suggest that soluble IrreC-rst dimerizes. Under the same conditions dimers of membrane bound full length protein could be shown as well.
Electron microscopic investigations on the localization of wild type and mutant IrreC-rst proteins
The intention of this diploma thesis was to find a method to examine the subcellular localisation of the cell adhesion molecule IrreC-rst by means of electron microscopy. This should e.g. allow the definite identification of the IrreC-rst positive cells in the developing retina and optic lobe. In a first step it was necessary to develop a fixation procedure which on one hand allows good immunoreactivity of the protein and on the other hand garantees a good tissue preservation. A method of DAB staining is described that has proven to be appropriate, although still some compromise had to be made with regard to tissue preservation. The examination of the eye imaginal disc from WTB and rstCT mutant flies showed localization of the protein in cell membranes of some retinula cells and in all (depending on timing) interommatidial and primary pigment cells. In the cytoplasm the protein was found in multivesicular bodies.
An interesting result is the different behaviour of the rstCT mutant protein (the intracellular domain of which is partially deleted) in neuronal and non-neuronal cells. In neurons the mutant protein is localised in the cell membrane (like in Wt B), but in non-neuronal cells it is found primarily in multivesicular bodies. This may indicate a faster degradation of the mutant protein in non-neuronal cells. The reason for the different behaviour in neurons is not yet known.
The localisation of the IrreC-rst protein in multivesicular bodies occurs in the eye imaginal disc and the optic lobe of Wt B flies as well. This reflects the dynamics of IrreC-rst expression pattern. Its spatial and temporal characteristics has been shown to be of functional importance (Schneider et al., 1995). In order to achieve such a highly regulated pattern, it is not enough to express the gene at a distinct time, the protein also has to disappear quickly at a given time. The internalisation and subsequent degradation of the protein represents such a mechanism for the elimination of a signal.
Characterization of regulatory regions of the irreC-rst gene
A. Straube, M. Struenkelnberg and E. Eule
An understanding of the highly regulated irreC-rst expression will enhance the understanding of the in vivo function of this gene, and also provide tools for time and tissue specific expression of transgenes in Drosophila. As a prerequisite we have to elaborate those regions of the irreC-rst gene which confer its developmental and tissue-specific expression pattern. We follow two different strategies.
First, we test fragments of 10 to 15 kb of the irreC-rst 5'-region in an in vivo enhancer tester systems. The fragments are being cloned in front of a basal promoter and a reporter gene and transformed into flies.
Second, we are cloning the D. virilis homologue of irreC-rst in order to compare coding and non-coding regions with those of D. melanogaster. As both species are 60 Mio years apart, we expect conservation of functional regions and dissimilarities in nonfunctional parts.
Does the BEN-protein, an avian axonal surface protein, function in Drosophila?
Zhiping Nie (in cooperation with Olivier Pourquié, Nogent sur Marne)
BEN ("bursal epithelium and neuron"), a 95 to 100 kd cell surface glycoprotein functioning during chicken development, is a homophilic member of the immunoglobulin superfamily composed of 5 extracellular immunoglobulin domains, a transmembrane region and a short intracellular tail. BEN is transiently expressed in various cell types, including neuronal, epithelial and hematopoietic cells. In the nervous system, BEN is expressed on axonal subtypes during growth and synaptogenesis. BEN is structurally related to IrreC-rst, a neural cell adhesion molecule of Drosophila that causes dominant axonal projection defects in when misexpressed by the Gal4/UAS system. We wondered whether similar effects could be caused by misexpression of BEN. Therefore a BEN cDNA was cloned into the pUAST vector and transformed into the germline of Drosophila. Crossing these transformants with Gal4 lines yielded F1 individuals displaying BEN immunoreactivity in a Gal4-line specific manner.
BEN misexpression does not mimick the effects of misexpression of IrreC-rst. In fact, no Gal4-line used so far has caused any mutant phenotype in combination with UAS-BEN, in spite of the fact that the BEN protein is transported into the axons of Drosophila and is able to mediate homophilic adhesion in vitro.
The conclusion is that BEN may require co-factors for functioning that are absent in Drosophila or at least in the cell types studied. One possibility is that the short intracellular domain of BEN is not sufficient to mediate intracellular interactions. For this reason we have constructed hybrid molecules between IrreC-rst and BEN that await testing.
The World Wide Web as a Tool for Scientific Communication
Karl-Friedrich Fischbach, Peter Robin Hiesinger and Armin Müller
The emergence of the internet is the most significant event in evolution at presence. We try to contribute by adding content that may enhance research and teaching efficiency.
FLYBRAIN is the database of the international Flybrain Project. The aim of the Flybrain project is to provide a schematic atlas of the Drosophila nervous system with click-on links to the real images. Educational tours around the fly's nervous system have been organized. Around this core, publication-like data will be assembled. Data entry will be peer reviewed by members of the editorial board. The data will be fully citable. Flybrain is run by a consortium (consisting of Drs. Nick Strausfeld, Tucson, Kei Ito, Tokyo and K.-F. Fischbach, Freiburg). In Freiburg FLYBRAIN is run on a server of the university library and supported by the DFG (grant to the library).
2. Permanent poster session of Drosophila neurobiology
Displaying a poster at a meeting for only a few hours is sometimes rather frustrating. Associated with the Flybrain project we offer Drosophilists a permanent exhibition space. In contrasts to the data entering the FLYBRAIN database directly, posters will not be peer reviewed. Their content is the responsibility of the authors alone. The status of poster informations is thus similar to that of posters at normal meetings. Citation as personal communication is possible after contacting the authors. We are working on a fully automated system for the entry of image and text information that will enable scientists around the world to enter their posters directly from their local computer.
3. Web Starting Points for Biologists
This is an emerging, hierarchically organized directory leading to information resources of putative interest for biologists (not only for Drosophilists). A special feature is that the subject lists can be extended by the users. Keyword search engines, databases, and educational sites for teachers and students are included.
4. Neurogenetik - Online
"Neurogenetik - Online" originated from a printed German teaching text. After translation into the hypertext markup language (HTML), it was realized that the incorporation of links pointing to external resources of biological information gives this text a new quality. Its usefulness increases daily by the growth of the remote sites. "Neurogenetik - Online" - can still be used in a conventional way, i.e. it can be read from Chapter 1 to the end, but it is also developing into a kind of organized directory for neurogenetic information from all around the world.
The "Neurogenetik - Online" project is one of 7 examples reviewed by the "Hochschul-Informations-System" (HIS) in their "Bestandsaufnahme zur Organisation medienunterstützter Lehre an Hochschulen - Fallstudien" (1996).
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