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First published online 14 December 2005
doi: 10.1242/dev.02191

Development 133, 275-285 (2006)
Published by The Company of Biologists 2006
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Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila

Heather L. D. Brown1,*, Lucy Cherbas2, Peter Cherbas2 and James W. Truman1

1 Department of Biology, University of Washington, Seattle WA 98195, USA.
2 Department of Biology and Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN 47405, USA.



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  		Fig. 1. Metamorphosis of the thoracic ventral (Tv) neurosecretory cells. (A) Schematic transverse section of the ventral nervous system, showing the axons, dendrites and cell bodies of a pair of Tv cells, before metamorphosis. The axons are wrapped around a pair of support cells (dorsal neurohemal organ), situated outside the neural sheath. (B) Projected confocal Z-stack of CD8::GFP expression in the thoracic neuromeres of an early metamorphic individual (18 hours apf) of the genotype UAS-CD8::GFP;FG10-GAL4. Expression in the thorax is limited to the three pairs of Tv neurons (arrows point to somata). (C) Dorsal view of the Tv cell axons, immunostained with {alpha}-SCP, at various time points during metamorphosis.

 


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  		Fig. 2. Z-stack projections from live imaging time-lapse videos, showing changes in the axonal arbors of Tv neurons. (A) Enlarged still of the most anterior axon arbor (T1) at 6.5 hours apf, showing filopodia extending from the neurohemal organ. (B) Z-stack time-lapse projection of the T2 (bottom) and T3 (top) neurohemal organs (arrows) from 21 to 29 hours apf, every 60 minutes. Pruning can be seen clearly as T2 decreases in size from 21 to 29 hours apf. Filopodia from the growth zone under each neurohemal organ fasciculate (arrowheads). The last panel is an enlarged still of the T3 neurohemal organ at 23.5 hours apf, showing the large number of filopodia. (C) Z-stack time-lapse projection of a pruned T3 axon arbor from 25 to 28 hours apf, showing the dramatic outgrowth that occurs between 25 and 28 hours apf.

 


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  		Fig. 3. Immunocytochemistry ({alpha}-SCP) of Tv cell axon arbors during the pruning phase of remodeling, for control cells and cells expressing EcR-DN. Variability in nervous system size and spacing of Tv cell neurohemal organs was seen in all treatments and did not appear to be correlated with any particular treatment. Inset: magnification of the T1 axon arbor at 24 hours apf of cells expressing EcR-DNs.

 


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  		Fig. 4. Z-stack projections from time-lapse videos of axons from Tv cells expressing EcR-B1F645A or EcR-B1W650A. (A) Z-stack time-lapse projection of two neurohemal organs (T2 above, T3 below) from cells expressing EcR-B1F645A from 20 to 23 hours apf, 60 minutes apart. No filopodia are visible during this time period. (B) Z-stack projections of remodeling axon arbors from cells expressing EcR-B1F645A. From 24 to 30 hours apf, filopodia from the partially pruned T1 (above) fasciculate and stabilize with filopodia from T2 (arrowhead). The last panel is an enlarged still of T1 at 26 hours apf, showing filopodia. (C) Z-stack time-lapse projections of axon arbors (T3 above, T2 below) from cells expressing EcR-B1W650A, from 23 to 29 hours apf, with an enlarged still at 26 hours apf, showing no filopodia.

 


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  		Fig. 5. Immunocytochemistry ({alpha}-SCP) of Tv cell axon arbors during the outgrowth phase of remodeling and quantification of arbor size during remodeling for control cells and cells expressing EcR-DN. (A) Immunocytochemistry ({alpha}-SCP) of Tv cell axon arbors during outgrowth, for control cells and cells expressing EcR-DN. (B) Comparison of arbor footprints from pupariation to adult. Arbor footprint was measured by taking a pixel count of the polygon created by connecting the outermost spread of the axon arbor.

 


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  		Fig. 6. Quantitative analysis of filopodial activity for Tv cell axons. Average filopodial number per hour for control cells and cells expressing EcR-DN was calculated from live imaging data for T1 (A), T2 (B) and T3 (C).

 


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  		Fig. 7. Quantification of EcR (common) and CD8 immunoreactivity in Tv cell bodies. (A) Cells expressing dominant negative, 24 hours apf. Inset shows the EcR to CD8 ratio for each genotype. (B) Tv cell bodies, stained with EcR (common) antibody (right) and CD8 antibody (left). (C) IR-EcR (core)-expressing cells, late third instar. Inset shows the EcR to CD8 ratio for each.

 


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  		Fig. 8. Expression of IR-EcR (core) in Tv cells during remodeling. (A) SCP-labeled, fixed Tv cell axons from cells expressing IR-EcR (core). (B) Arbor footprint, from pupariation to adult for control cells and cells expressing IR-EcR (core). (C) Live imaging montage of Tv axons (T1, T2 and T3; T1 at top) of UAS-IR-EcR (core)-expressing cells, from 32 to 39 hours apf, 60 minutes apart. Filopodia are present and stabilize into adult-type branches (arrowhead), but the arbor retains a larval-like dense core (arrow). (D) Quantification of filopodia on control cells and cells expressing IR-EcR (core).

 


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  		Fig. 9. Conclusions. Model of EcR action and summary of the effects of EcR dominant negative and RNAi treatments during the pruning and sprouting phases of the Tv cell axons.

 

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© The Company of Biologists Ltd 2006