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doi: 10.1242/10.1242/dev.00598

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Targeted expression of shibire^ts and semaphorin 1a reveals critical periods for synapse formation in the giant fiber of Drosophila

¹ University of Massachusetts, Department of Biology, Morrill Science Center, Amherst, MA 01003, USA
² Division of Neurosciences, Beckman Research Institute of the City of Hope, 1450 E.Duarte Road, Duarte, CA 91010, USA

View larger version (63K): [in a new window]   Fig. 1. Development of the GF system. (A) GF development. The timing of events is based on the work presented in the present paper as well as the dye injection work of Jacobs et al. (Jacobs et al., 2000). The time line is recorded as percent of pupal development with time zero according to Bainbridge and Bounds (Bainbridge and Bounds, 1981). The phases indicated below the time line were determined experimentally in the present work. The time of dye coupling was determined by Jacobs et al. (Jacobs et al., 2000). t1, t2 and t3: first, second and third thoracic neuromere. (B) Expression patterns of the three Gal4 constructs. (B1) Simultaneous staining of the GF (with c17) and the TTMn (with shakB-Gal4) at 33% of pupal development. In this particular specimen, the left GF has reached the TTMn and the right GF has not. (B2) When driven by shakB-Gal4, lacZ is expressed in the postsynaptic motoneurons and TTMn exhibits the strongest staining. (B3) The P[Gal4]a307 enhancer is expressed strongly in the GF and more weakly in the postsynaptic cells. In this specimen both GFs are labeled and the dendrites of the left TTMn are revealed. (B4) The P[Gal4]-c17 enhancer is expressed in the GF but not in the motoneurons. The developmental stage of each specimen is indicated in the lower right of each panel. Scale bar: 20 µm.

View larger version (59K): [in a new window]   Fig. 2. Defects in the adult GF and TTMn are induced by blocking endocytosis at various stages in development. (A) Anatomy of the GF system after various temperature shifts when UAS-shits was driven by the A307 Gal4 enhancer. (A1) A temperature shift at 16% of pupal development followed by regeneration and examination in the adult stage. Two specimens are represented. In one a GF is trapped in the brain, in the thorax of another both GFs exhibit the `overgrowth' phenotype. (A2) A temperature shift at 33% of pupal development produced a bendless-like phenotype where the large lateral bend is missing from both GFs in the adult. (A3) A temperature shift at 75% of development had no detectable effect on the structure of the GF. (A4) Both late temperature shifts and controls exhibit normal bends. This particular specimen was never temperature shifted and illustrates the structure of the GF in the adult stage. Scale bar: 20 µm. (B) Physiology of the GF system. Each pair of traces is taken from a specimen temperature shifted at the time indicated. (B1) Early temperature shifts disrupted the TTM muscle and no recordings could be obtained. The DLM was often excited by the GF but latencies were long and very few stimuli in a train elicit a response. (B2) Temperature shifts during synapse formation increased the latencies and decreased following frequency. (B3) Response latencies were increased and, following frequency, decreased when temperature shifts occurred between 62.5-75% of pupal development. (B4) Temperature shifted at 84% had no statistically significant effect on the physiology and this specimen illustrates the normal physiology (see Table 1 for quantification). In control specimens, the latency for TTM is about 0.9 mseconds and for DLM was 1.4 mseconds; both motoneurons could follow the 100 Hz stimulus without fail. The upper trace in each panel is taken from the TTM, the lower trace from the DLM. In each set of traces, the individual stimulus illustrates the latency and wave form of the response, the sweep with 10 stimuli illustrates the response to repetitive stimuli.

View larger version (93K): [in a new window]   Fig. 3. The dynamics of axon retraction and regeneration. Each row represents the results for a temperature shift initiated at the time indicated. Each column represents the structure at a different time with respect to the temperature shift. The left column shows examples immediately before the temperature shift, the middle column immediately after the temperature shift and the right column illustrates the adult CNS after the temperature shift. (A) Temperature shift initiated at the beginning of pupariation (P0). (A1) Schematic version of the GF at P0. The GF has not reached the target area in the early phase of growth and the axons are very thin, making it difficult to stain and visualize with our methods. The schematic illustrates the approximate location of the axons at this time as well as showing the portion of the CNS represented in all the panels (dashed box). (A2) The axons at the end of a temperature shift that began at pupariation. Both axons have retracted into the anterior end of the thorax and each exhibits a retraction bulb (arrow) near the terminal and a thin retraction tail extending toward the target. (A3) Overgrowth of the axon in the adult after regeneration at the permissive temperature. (B) Temperature shift initiated at 33% of pupal development (Phase II). (B1) The structure of the GF at 33% of development. Most specimens exhibited the laterally projecting terminal illustrated by the left GF, a minority of GFs have just begun to make the bend as seen for the right GF. (B2) Dissection at the end of the temperature shift (50%) shows that both axon terminals have retracted into the first thoracic neuromere and terminate in retraction bulbs (arrow). In this specimen the dendrites of the TTMn were also visible (arrowheads) indicating the extent of the GF retraction. The dendrites of the TTMn appeared normal in this specimen. (B3) After regeneration, both GFs tapered to an end in the target area and neither showed the laterally projecting terminal. (C) Temperature shift initiated at 50% of pupal development. (C1) Both GFs exhibited the normal lateral extension of the presynaptic terminal at 50% of pupal development. (C2) Retraction of the presynaptic terminal immediately after a temperature shift. Note that the presynaptic terminal has withdrawn but the axon did not retract away from the target area. (C3) The adult regenerated axon. In this case a large swollen region was present on the right GF just anterior to the presynaptic terminal. (D) Temperature shift initiated at 66% of pupal development. (D1) The GF structure at 66% of pupal development exhibited normal lateral extensions. (D2) Immediately after a temperature shift, there was no obvious defect. (D3) An adult specimen indistinguishable from controls. Genotype of all specimens; UAS-lacZ/+;A307/+;UAS-shits/+. Scale bar: 20 µm.

View larger version (148K): [in a new window]   Fig. 4. Interactions between shits and sema1a. Each row illustrates the results for a different UAS-construct, driven by c17. Each column indicates the results for a temperature shift at the indicated time. All specimens were dissected in the adult stage. (A) Specimens expressing UAS-sema1a alone. (A1,A2) Temperature shifts during synapse formation caused bendless-like phenotypes. (A3) Late temperature shifts (62.5% of pupal development) had no effect and were indistinguishable from (A4) controls that were not temperature shifted. (B) Specimens expressing only UAS-shits. Very few defects were observed, usually all axons reached the target area and extended a lateral bend independent of the temperature shift. (C) UAS-sema1a and UAS-shits were targeted to the presynaptic GFs. (C1) When temperature shifted at 37.5% of pupal development GFs exhibited a swollen terminal filled with vesicles in the target area (arrow). (C2) The left GF in this example at 50% of pupal development exhibits a swollen terminal and a retraction tail, the right GF is dramatically swollen and filled with membrane bound vesicles (arrow). (C3) Late temperature shifts cause some retraction and few swellings or vesicles. (C4) Control specimen that was not temperature shifted. Scale bar in A1: 20 µm.