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First published online 20 July 2005
doi: 10.1242/dev.01928

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Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons

Darren W. Williams^*, and James W. Truman

Department of Biology, University of Washington, Seattle, WA 98195, USA

View larger version (123K): [in a new window]   Fig. 1. Deconstruction of the abdominal sensory system during early metamorphosis. Projected z-stacks of neurons labeled by C161-GAL4>CD8::GFP in the dorsal abdomen from (A) a wandering third instar larva (wL3). Neurons labeled include the dorsal multiple dendrite neuron (dmd), dorsal bipolar dendrite neuron (dbd) and five dorsal dendritic arborizing neurons (ddaD, ddaE, ddaF, ddaA and ddaB), those labeled in white survive. The inset shows merged projected z-stacks labeled with anti-Cut (magenta) and anti-CD8 (green) to allow identification of neurons. The sixth dorsal da neuron, ddaC is weakly labeled. Scale bar: 90 µm. (B) By 6 h APF the arbors of ddaF, ddaB and ddaA have largely disappeared (yellow arrow). The cell bodies of dead cells are found close to the nerve (yellow arrowhead). The dendrites of ddaD are still intact. Box denotes region imaged in Fig. 2A. Scale bar: 40 µm. (C) At 10 h APF ddaD shows signs of pruning. (D) At 16 h APF the higher order branches are gone and detached branches are found near the arbor (arrowhead). The proximal branch is thinning. (E) At 24 h APF only ddaD, ddaE and dbd remain. Note filopodia on pruning branches (white arrows). Dorsal is up and anterior to the left.

View larger version (119K): [in a new window]   Fig. 2. Dynamics of dying da neuron dendrites. (A) Frames from Movie 1 (see supplementary material) of wild-type neurons (C161-GAL4>CD8::GFP) in the dorsal region of the prepupa (boxed region in Fig. 1B). The drawing shows distal arbors of ddaF (yellow), ddaB (green) and ddaD (blue) at 0 h APF. At 0 h arbors have a uniform caliber indistinguishable from those of the larva. By 2 h APF the dorsal branch of ddaF develops constrictions and swellings (yellow arrows). By 3 h APF the swellings become bead-like (yellow arrows). Between 3 and 4 h APF the branch severs, leaving blebs of GFP. The branches of ddaB generate swellings (green arrows). Between 4 and 6 h they sever, and the GFP blebs rapidly disappear, leaving only ddaD intact (blue arrow). Scale bar: 20 µm. (B) Projected z-stacks of ddaF in a wild-type animal (C161-GAL4>CD8::GFP). Cell bodies and proximal arbor (p) are shown on the left with the distal (d) dorsally projecting arbors on the right. At 1 h 30 min a distinct bead forms at a proximal branch point (arrow), whereas the most distal part (to the right) is intact. By 3 h the distal region is completely beaded. At 4 h the beads have moved position. Scale bar: 25 µm. (C) Projected z-stack of dorsal abdomen of wild-type (C161-GAL4> tub::GFP) at 3 h APF. The distal and proximal regions (boxed) are shown in the panels on the right. The distal arbor of ddaF has distinct beads (yellow arrows) whereas ddaD has a uniform distribution of tub::GFP (blue arrow). The nucleus and the proximal branches of ddaD and ddaE are intact, next to remains of ddaF (yellow arrowhead) and ddaB (green arrowhead). Dorsal is up and anterior to the left. Scale bar: 40 µm. (D) p35 inhibits cell death in all neurons expressing GFP and p35 at 5 h APF (C161-GAL4>CD8::GFP, p35). Left panel shows intact cell bodies; arrowheads indicate ddaF and ddaA (yellow), ddaB, (green), and ddaD and ddaE (blue). The arbor of ddaD (blue arrow) would normally be the only one left by this time in development. Right panel shows distal arbors of the same cells. Scale bar: 30 µm. (E) At 18 h APF all neurons are still alive in C161-GAL4 animals expressing p35. Arrow indicates intact primary branches of ddaB and ddaF. Scale bar: 30 µm.

View larger version (140K): [in a new window]   Fig. 3. The dynamics of dendritic pruning. Projections of z-stacks taken from movies of a wild-type pupae (C161-GAL4>CD8::GFP). (A) Overview of Movie 2 (see supplementary material). At 14 h 10 min APF as the arbors of ddaD and ddaE are pruning, fragments and blebs of GFP are found close to the arbor (arrowheads). Filopodia extend on ddaD's primary branch (arrows indicate sites of future severing). By 17 h 10 min ddaD has severed at two sites (arrows). The primary branch of ddaE is thinned (arrowhead). Asterisk indicates where arbor temporarily moved out of the z-stack. By 19 h 00 min the primary branches of both neurons have been separated from the cell body. Filopodia extend from the cell bodies (arrow). Between 19-24 h the severed branches decrease in length, thin (arrowhead) and generate swellings (arrow). Scale bar: 40 µm. (B) Proximal thinning of dendrites. Selected frames from Movie 2 focusing on a proximal region of ddaD and ddaE. Arrowheads indicate beads. Filopodia extend and retract on the thinning branch throughout the sequence (arrows, 17 h 00 min). Between 17 h 00 min and 17 h 50 min ddaE's primary branch becomes noticeably thinner. Just prior to 18 h ddaE is severed, isolating the distal arbor. (C) Distal branch severing. Overview of distal severing in ddaD; box indicates area enlarged in the panels on the right. At 14 h 10 min beads are present at the site labeled with the arrow. The beads are dynamic and change position during the movie. At 15 h 10 min there is no obvious thinning at the site where the arbor will sever (arrowhead) Branch labeled with asterisk retracts a short distance following severing. Scale bar: 20 µm. (D) Branch fragmentation. Branch severed from the primary branch (lower arrow in A) shows thinning at the ends by 17 h 50 min. At 18 h 50 min the branch begins to fragment into blebs, and by 20 h 00 min the only trace of the branch are a few blebs of GFP remaining in the field. Arrow indicates beads; arrowhead denotes thinning. Scale bar: 40 µm. (E) Branch retraction. Frames from a movie showing a retraction event on the primary branches of ddaD. The primary branch shows dynamic filopodia (arrowhead) along its length. Retraction is evident at 18 h 30 min and is back to it cell body by 21 h 00 min. The ventral branch to the left undergoes fragmentation (arrow). Scale bar: 35 µm.

View larger version (82K): [in a new window]   Fig. 4. Cytoskeletal remodeling of dendrites during early metamorphosis. (A) Projected confocal z-stack of ddaC in the abdomen of a wild-type larva (ppk1.9GAL4> CD8::GFP). The box denotes the region from which images B-C' were collected. Scale bar: 30 µm. (B,B') The proximal branch of ddaC at wandering third instar larva stage labeled with anti-CD8 (B) and anti-Futsch (B'). Arrowhead indicates branches from another da neuron. (C,C') The proximal branch of ddaC at 3 h APF labeled with anti-CD8 (C), anti-Futsch (C'); arrows denote beads on branches. Filopodia (f) are found on branches that have undergone thinning; arrowhead indicates another da neuron. Scale bar: 10 µm. (D) Dynamics of the microtubule cytoskeleton of ddaC during the first six hours of metamorphosis. Projections of z-stacks from a movie showing tub::GFP distribution. 0 h APF reveals the uniform distribution of tub::GFP. By 2 h APF tubulin is redistributed resulting in a decrease in some places and an increase (arrow) in others. By 4 h APF some regions lose significant quantities of tub::GFP while others accumulate it into beads (arrow). By 6 h APF very large beads have formed that are separated by lengths of arbor that have little to no GFP signal. Scale bar: 30 µm.

View larger version (90K): [in a new window]   Fig. 5. Blocking ecdysone signaling in da neurons with EcRDN. (A) Projected z-stack of wild-type neurons (C161-GAL4>CD8::GFP) in the dorsal abdomen at 16 h APF. (B) Projected z-stack of neurons expressing EcRDN (C161-GAL4>EcRDN; CD8::GFP) in the dorsal abdomen at 16 h APF. No severed branches are present and only a few blebs of GFP are evident. Arrows indicate distal dendrites of ddaD and ddaE, arrowheads the distal dendrites of ddaF. Scale bar: 20 µm. (C) The proximal branches of dorsal da neurons in a wild-type animal at 16 h APF are thinned. Scale bar: 7 µm. (D) Equivalent proximal branches of dorsal da neurons expressing EcRDN at 16 h APF show no thinning. Asterisk indicates cell bodies of ddaF and ddaB still present. (E) Distal region of branch in a wild-type animal at 16 h APF. Arrows indicate fragments. (F) Distal region of ddaD expressing EcRDN at 16 h APF. Arrow indicates retraction bulb. (G) Dynamics of the pruning of dorsal da neurons expressing EcRDN during early metamorphosis. Arrows indicate branch retraction. At 24 h APF the dendrite branches are shorter and the retraction bulbs have increased in size. Branches continue to retract (arrows). Arrowhead in 25 h 30 min and 26 h 40 min identifies a distal severing event. Scale bar: 20 µm (16 h APF); 25 µm (other times).

View larger version (67K): [in a new window]   Fig. 6. Remodeling of the abdominal epidermis. (A) The position of ddaD and ddaE (yellow arrow/arrowhead) in relation to the larval epidermal cells (pink arrows) and smaller diploid cells of the histoblast nests (pink outline) at 5 h APF in a H2Av::GFP, C161-GAL4>CD8::GFP animal. Scale bar: 45 µm. (B) Removal of larval epidermal cells by phagocytes. Projections of z-stacks from a movie with H2Av::GFP labeling the nuclei of all cells. The boxed region shows the position of the other enlarged images relative to the imaginal histoblast nest (IHN). At 15 h 00 min the larval epidermal cell nucleus is intact (pink arrow), and the small diploid nuclei of phagocytic blood cells (white arrows) are found close by. Between 16 h 40 min and 17 h 20 min the nucleus moves and changes shape, identifying that it has been consumed by a phagocyte. For the remainder of the movie the remnants of the nucleus can be seen inside the phagocyte. Scale bar: 35 µm. (C) Fate of 106 larval epidermal cells during early metamorphosis (14 and 25 h APF). During this time period, 17 larval epidermal cells died (orange dots). Scale bar: 50 µm.

View larger version (166K): [in a new window]   Fig. 7. Phagocytic blood cells engulf neuronal debris and attack intact dendrites during pruning. (A) Frames from a time-lapse movie of da neurons (C161-GAL4>CD8::GFP) showing phagocytic blood cell consuming a detached dendrite branch. At 19 h 10 min a weakly labeled phagocyte enters from the right (arrowhead). By 19 h 40 min the phagocyte has moved close to the severed branch. At 19 h 50 min the branch fragments and by 20 h 50 min the phagocyte, is strongly labeled with GFP. Scale bar: 20 µm. (B) A projected z-stack of a phagocyte labeled with LysoTracker DND-99 in a wild-type animal (C161-GAL4>CD8::GFP) showing details of vesicles. Different sized vesicular compartments that do not contain GFP are evident (arrow) along with ones that do (arrowhead). Scale bar: 7 µm. (C) Phagocyte attacking distal tip of ddaE. Selected frames from a time-lapse movie. At 16 h 00 min a phagocyte can be seen on the right which then comes into contact with the distal tip of the branch (top arrowhead). By 17 h 00 min the branch and the phagocyte are no longer in contact and the phagocyte contains more GFP than before. Another phagocyte crosses the branch from the lower left (arrowhead) attacking the retracting end of the primary branch between 19 h 10 min and 20 h 30 min. Scale bar: 35 µm. (D) Phagocytic blood cells attack intact proximal branches. At 15 h 40 min a phagocyte enters the field from the left. Between 15 h 40 min and 17 h 30 min it moves close to the pruning arbor of ddaE (arrowhead). Between 18 h 10 min and 18 h 20 min the branch is severed at the site where the phagocyte is located. A small bleb of GFP, derived from the severed branch is internalized by the phagocyte and moves away with the blood cell (18 h 30 min arrowhead). Scale bar: 25 µm.

View larger version (119K): [in a new window]   Fig. 8. Phagocyte behavior changes when pruning is blocked. False color indicates the different frames of the respective time-lapse movies; magenta, the first frame and green the final frame. The path of a phagocyte through all of the frames is recorded as a white line, and the final destination denoted with an arrow. (A) In a movie of a wild-type animal (C161-GAL4>CD8::GFP) starting at 0 h (14 h APF) the phagocyte tracks around the ventral branch of ddaD until it is severed and then moves to the right attacking the retracting branch of ddaE. (B) In a movie of EcRDN expressing neurons (C161-GAL4>EcRDN;CD8::GFP) starting at 0 h (14 h 20 min APF) two phagocytes have been tracked. The phagocyte to the left follows the distal tip of the branch, removing pieces as it retracts. The other phagocyte is found near the proximal arbor and moves slowly across the field and does not interact with the `non-thinned' branches. Scale bar: 30 µm.

View larger version (26K): [in a new window]   Fig. 9. Summary of cellular mechanisms of da neuron dendrite pruning.