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First published online 21 July 2004
doi: 10.1242/dev.01274

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Sra-1 interacts with Kette and Wasp and is required for neuronal and bristle development in Drosophila

Institut für Neurobiologie, Universität Münster, Badestrasse 9, Münster, 48149, Germany

View larger version (53K): [in a new window]   Fig. 1. Sra-1 binds Kette through its C terminus. (A) Whole-mount stage 16 embryos stained for kette or sra-1 RNA expression as indicated, (Left) Lateral views; (middle) ventral views; (right) higher magnifications of the ventral nerve cord showing ubiquitous expression of both genes. (B) Western blot analyses of Kette (120 kDa) and Sra-1 (140 kDa) expression. Both proteins are expressed throughout embryogenesis and appear to be provided maternally. (C) Co-immunoprecipitation of Kette and Sra-1. Total cell lysate of S2-cells expressing both Myc-tagged Kette and Sra-1 was subjected to immunoprecipitation with monoclonal anti-Myc (9E10) or anti-Slit (E555.6D). Sra-1 is detected only in the anti-Myc precipitate, indicating binding of Kette and Sra-1. (D) To test the interaction between Sra-1 and Kette, we turned to a yeast two hybrid assay. Interaction between Sra-1 and Kette results in growth of the yeast cells and a blue color. Interaction is seen only when the C terminus is intact (E).

View larger version (67K): [in a new window]   Fig. 3. sra-1 is required for axonal growth. Confocal images of mount preparations of third instar eye discs (ed) for Sra-1 expression (green) and the axonal marker 24B10 (red). The merge channel is shown in the bottom row. (A) In wild type, photoreceptor neurons express both Sra-1 and 24B10, and project their axons through the optic stalk (os) to the lamina (la) or medulla (me). (B) Following expression of sra-1 dsRNA using an eyeless-GAL4 driver, Sra-1 protein expression was removed from the eye disc but not the brain (star). Concomitantly, we observed severe axonal targeting defects. Most axons appear to be able to enter the brain, but fail to grow towards their correct targets (arrow). (C,D) Adult eyes of (C) a wild-type fly and (D) a transgenic fly expressing sra-1 RNAi in the eyeless pattern.

View larger version (74K): [in a new window]   Fig. 5. Kette and Sra-1 stabilize each other. (A-C) Top row, expression of Kette (green) in S2R+ cells; bottom row, F-actin was detected using phalloidin (red). (A) Wild-type S2R+ cells grown on an adhesive substrate. (B) Kette expression in S2R+ cells can be suppressed by treatment with double-stranded kette RNA (RNAi). Retraction fibers and clumps of F-actin are found within the cells (arrowheads). (C) When sra-1 function is inhibited by sra-1 RNAi, Kette protein expression cannot be detected after 2 days of RNAi treatment. In addition, a F-actin phenotype develops (compare B with C). (D) Western blot analysis showing mutual protein stabilization of Kette and Sra-1. sra-1 RNAi affects Sra-1 and Kette expression and, conversely, kette RNAi efficiently reduces Kette and Sra-1 protein expression.

View larger version (110K): [in a new window]   Fig. 2. Neuronal expression of Sra-1. Confocal images of mount preparations for Sra-1 expression (green) and axonal markers 24B10 or BP102 (red). The merge channel is shown on the right. (A) During embryogenesis Sra-1 (green) accumulates in the neuropile of the central nervous system, which also expresses the BP102 epitope. (B) In the optic ganglia of third instar larval brains Sra-1 protein expression can be detected in the medulla (md) and photoreceptor axons that traverse the lamina (la). An intense label can be seen in the termination zone of the photoreceptor axons R1-R6 in the lamina (star). The growth cones of these axons also express high levels of the 24B10 antigen (red). (C) Cell bodies of the photoreceptor neurons also express Sra-1, albeit at lower levels (arrowhead). ed, eye imaginal disc; os, optic stalk.

View larger version (33K): [in a new window]   Fig. 4. kette and sra-1 affect synaptic development. Neuromuscular junctions (NMJ) of muscle 4 taken from third instar larvae are shown. Boutons are labeled by Nc46 expression. The inlay shows a higher magnification of a terminal bouton highlighted by the boxed area. (A) Wild-type NMJ. Note the smooth morphology of the terminal boutons. Almost no extra branches or buds can be detected. (B) After sra-1 RNA interference, an increased budding of terminal boutons was noted (elavGal4xUASsra-1RNAi). (C) Third instar larvae carrying the weak hypomorphic allele combination ketteJ1-70/kette26. There is an increase in budding tendency, which is particularly evident at the terminal boutons. (D) A similar phenotype was observed following neuronal expression of the sra-1CMyr deletion construct (elavGal4xUASsra-1CMyr).

View larger version (74K): [in a new window]   Fig. 6. kette and sra-1 function during bristle development. (A-C) Top row, dorsal view of Drosophila heads; bottom row, higher magnification showing the morphology of macrochaetes. (A) Hypomorphic kette mutant flies (ketteJ1-70/kette2-6) are characterized by bent bristles (arrow). (B) Following expression of sra-1 dsRNA in the scabrous pattern, similar bent bristles are formed (arrow). Some bristles are missing (star). This phenotypic trait increases following expression of higher levels of Sra-1 RNAi. (C) After expression of a membrane-tethered Sra-1 protein variant lacking the Kette interaction domain (Sra-1CMyr), frequent loss of bristles as well as bent bristles is observed.

View larger version (197K): [in a new window]   Fig. 7. Sra-1 affects F-actin formation. During pupal development, bristle morphology is prefigured by an apical F-actin extension, which can be visualized by phalloidin staining. All pupae were dissected 36 hours after puparium formation. (A) Wild type. (B) After expression of sra-1, dsRNA expression (one copy) using sca-GAL4 bent F-actin bundles, which are never seen in wild type, can be detected. (C) After expression of one copy of the sra-1CMyr construct, no F-actin forms at the site where bristles are expected to develop (star). (D) Following expression of full-length membrane tethered Sra-1 protein using the sca-GAL4 driver, F-actin formation is initiated in a broader region of the apical cell surface (arrow). In addition, the F-actin at the cell boundary appears to have a `fuzzier' organization (arrowheads).

View larger version (47K): [in a new window]   Fig. 8. Genetic interaction between sra-1, kette and wasp. (A-H) Morphology of microchaete on the thorax in different genetic backgrounds. Quantitative analyses are shown in I. (A) Wild-type flies are characterized by an ordered array of microchaete, which are normally thin, straight and have a pointed end. (B) Following expression of one copy of the UAS-Sra-1Myr transgene in the scabrous pattern, shorter and split bristles develop (arrow). (C) The same phenotype is observed after expression of membrane tethered Kette protein (arrow). (D) Co-expression of both membrane-tethered Sra-1 and Kette leads to a synergistic increase of bristle defects. (E) Same genetic background as in B but lacking one copy of the kette gene. The bristle phenotype evoked by Sra-1Myr expression is not affected. (F) Same genetic background as in B but lacking one copy of the wasp gene. The bristle phenotype evoked by Sra-1Myr expression is suppressed. (G) Expression of a UAS-Sra-1CMyr transgene in the scabrous pattern results in loss of bristles. (H) When flies co-express UAS-Sra-1CMyr and UAS-KetteMyr transgenes, the loss of bristle phenotype is suppressed. However, bristles are brushed, which corresponds to the KetteMyr phenotype. (I) Quantitative analyses of the above-mentioned phenotypes. Threehundred microchaete were counted in each case.