Characterization of Gal4 Lines with Expression
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Annette Sulzbacher, Christian Reiter & Karl-Friedrich Fischbach
Institut für Biologie III, Schänzlestrasse 1, 79104 Freiburg i. Br., Germany
Results & Discussion
We have used the Gal4/UAS targeted expression system to analyze the regional specificity and developmental dynamics of reporter gene expression in a collection of enhancer trap lines. These lines express the yeast transcription factor Gal4 in subsets of cells within the optic lobe of Drosophila. As a reporter, we used a tau-GFP fusion construct (1). This fusion protein is transported into axonal processes due to the microtubuli-binding properties of the tau protein. It offers a high level of resolution in structural analysis. Due to its natural fluorescence, it is ideally suited for combination with antibody stains for two-channel confocal laser scan microscopy.
We have used tau-GFP to analyze expression patterns in four Gal4 lines (Mz507, Mz1407, Mz1369, Mz 1525, all obtained from J. Urban and G.M. Technau, Mainz) that produce axonal projection phenotypes when crossed to UAS-irreC-rst (2). Two of these lines (Mz1525, MZ1407) show projection defects mostly restricted to the inner chiasm, while in the others (Mz507, Mz1369), both optic chiasms develop abnormally. Comparison of the phenotypes seen in UAS-irreC-rst transformants with the expression patterns revealed with UAS-tau-GFP allows to test experimentally models for the action of IrreC-rst in optic chiasm formation. Double staining experiments stress the fact that IrreC-rst marks young axon bundles during optic chiasm formation (see Fig.6). Adult serial sections and larval stages of tau-GFP-expressing animals were also examined to ensure that no developmental defects are induced by the fusion protein. We observed that, in contrast to earlier investigations on similar reporter fusions (3), the tau fragment seems to be biologically active in Drosophila and interferes with neuronal differentiation and axonal pathfinding. Two of four investigated lines showed defects that we judge to be tau-mediated. Effects of tau-GFP are similar, though less severe, to those of a UAS-tau construct we used as a control. Mz507/UAS-tau-GFP animals were viable as adults but showed irregular agglomerations of axons in the lamina. In Mz1369 tau-GFP expression as well as tau expression, proved lethal. In the optic lobe the photoreceptor differentiation was inhibited and the lamina did not develop. Animals did not develop beyond the early pupal stage.
Fig.1: Larval optic lobe of Gal4 1407/UAS-tau-GFP . Fig.1A : Horizontal section. The IrreC-rst protein is localized (red) in the lamina (la), medulla (me), lobula (lo) and in the youngest fibres of the outer (x1) and inner (x2) chiasma. This typical IrreC-rst expression is used in the following pictures as a "map" to localize the specific structures of different Gal4 lines. In this line tau-GFP (green) is expressed in the medulla cortex (mc), the transient neuropil (tN), medulla, lobula, lobula cortex (lc), T/C cells and older fibres of the outer and inner chiasma. Scale bar 10mm, anterior is left. Fig.1B: Corresponding frontal section. The neuropils of the lamina and medulla form a crescent around the lobula plate cortex. The fibres of the T/C cells are placed directly adjacent to the lamina and project into the medulla.
Fig.2 : Gal4 1407/UAS-tau-GFP. Series of sagittal optic slices. A, B and C are progressively deeper slices from the optic lobe. Scale bar 16mm.
Fig.3: Gal4 1407/UAS-tau-GFP. Series of oblique optic slices showing projections in the inner chiasm and of the T/C cells. Scale bar 14mm. These images demonstrate that tau-GFP in combination with reference markers is suitable for a detailed three-dimensional reconstruction of expression patterns in the Drosophila nervous system.
Fig.4: Gal4 1525/UAS-tau-GFP. Sagittal section. GFP expression is between medulla, lobula and the region of the inner optic chiasm. Expression in the lobula complex is complementary to Gal4 1407 (see Fig. 2).
Fig.5: Gal4 1407/UAS-lacZ. Frontal section. DTAF anti b-galactosidase in green and IrreC-rst in red. b-galactosidase is not localized along the axons of the C/T cells compared to Fig.3 A where the T/C cells are stained by tau-GFP.
Fig.6: Gal4 507/UAS-tau-GFP. Horizontal section. This larval optic lobee exxpresses GFP (green) in the older fibers of both optic chiasms. Normal IrreC-rst expression (red) is complementary in the chiasms; it labels young fibre vundles and is downregulated on older ones. On fibre terminals in the neuropile IrreC-rst expression persists into the pupal stage. Colocalization of GFP and IrreC-rst results in the yellow colour.
Fig.7: Frontal paraffin sections (adult). A: Wildtype. The lamina (la) is devided into cortex and neuropil. B: Gal4 507/ UAS-tau-GFP. The white arrow points to an agglomeration between the lamina cortex and the lamina neuropil caused by tau-GFP expression.
Fig.8: Horizontal paraffin sections (adult). A&B: Gal4 507/UAS-tau. The structure of the optic lobe is defect. Lamina (black asterisk) and medulla are not visible as seperate neuropils. Scale bar 60 mm. C: Wildtype. Normal organisation of the optic lobe with lamina (la), medulla (me), lobula (lo) and lobula plate (lp).
These results show that, although tau fusion proteins offer a good resolution of structural analysis in the developing nervous system, lines used have to be carefully checked for possible defects caused by the fusion protein. Expression patterns should be closely compared to those seen with various reporter or effector constructs. However, studies using Drosophila expression systems may also offer novel insights into the functional capabilities of proteins such as tau.
Key words: Visual system - GFP - enhancer trap technique.
Brand, A. (1995): GFP in Drosohpila. Trends in Genetics 11, 324-325.
Schneider, T., Reiter, C., Eule, E., Bader, B., Lichte, B., Nie, Z., Schimansky, T., Ramos, R.G.P. & Fischbach, K.-F. (1995): Restricted expression of the IrreC-rst protein is required for normal axonal projections of columnar visual neurons. Neuron 15, 259-271.
Callahan, C.A. & Thomas, J.B. (1994): Tau-b-galactosidase, an axonal targeted fusion protein. Proc. Natl. Acad. Sci. USA 91, 5972-5976.
We thank Juan Botas and Immaculata Canal for providing the UAS-tau-gfp strain and Joachim Urban and Gerhard Technau for Gal4-lines.
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