The growth of Drosophila bristles and laterals is not restricted to the tip or base

Xiaoyin Fei*, Biao He{ddagger} and Paul N. Adler§

Biology Department and Cancer Center, University of Virginia, Charlottesville, VA 22903, USA
* Present address: University of North Carolina Medical School, Chapel Hill, NC 27514, USA
{ddagger} Present address: Thoracic Oncology Laboratory, UCSF Cancer Center, University of Califonia, San Francisco, CA 94115, USA

§ Author for correspondence (e-mail: pna{at}virginia.edu)

Accepted 24 July 2002


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The highly elongated bristles of Drosophila have proven to be a valuable model system for studying cellular morphogenesis. Extending bristles contain a series of large bundles of actin filaments juxtaposed to the plasma membrane and centrally located microtubules. Models to explain the extension of the bristle have principally focused on the assembly of actin filaments at the distal tip of the bristle. We have used time-lapse observations of wild-type and mutant bristles and the related arista laterals and come to the conclusion that growth takes place throughout the growing cellular extension. This distributed growth can explain the behavior of split laterals and the shape changes seen at the tip during bristle and lateral outgrowth. Inhibitor studies suggest that the microtubule cytoskeleton is essential for maintaining the highly biased axial growth of these structures. We have used fluorescence recovery after photo-bleaching to study the dynamics of the cytoskeleton during bristle growth. Our experiments show that actin bundles in growing bristles are quite stable and move in a retrograde fashion. The bristle microtubules are less stable. The retrograde movement of the peripheral actin appears to be counterbalanced by the distally directed movement of cytoplasm in the center of the bristle.

Key words: Cytoskeleton, Drosophila, Bristles, Outgrowth


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dramatically polarized cells are valuable models for the regulation of cellular morphology as their extreme shape puts a high demand on the cytoskeleton and makes variation easy to detect. The sensory bristles that decorate the adult cuticular landscape of Drosophila have long been a favorite system for such studies (Lees and Waddington, 1942Go; Tilney et al., 1995Go). These large cells are easily seen in the stereomicroscope, which has facilitated isolating mutations that alter bristle morphology. Developing bristles contain large bundles of actin filaments juxtaposed to the plasma membrane (in the thoracic microchaetae there are 7-11 bundles) and a large number of microtubules evenly distributed across the center of the developing shaft (Tilney et al., 1995Go). We have recently begun to use the lateral side branches of the arista as a complementary highly polarized cellular extension (He and Adler, 2001Go; He and Adler, 2002bGo). The arista is comprised of a thin multicellular cylinder (referred to as the central core) and a series of very thin lateral extensions. The laterals are produced as outgrowths from the apical surface of a small number of central core cells. In thin cross sections laterals are essentially indistinguishable from bristles, as they also contain large bundles of actin filaments juxtaposed to the plasma membrane and centrally located microtubules (He and Adler, 2001Go). Epidermal hairs are a third type of cellular structure that has served as a model in Drosophila (Wong and Adler, 1993Go).

There is both pharmacological and genetic data showing the importance of the actin cytoskeleton for bristle, lateral and hair morphogenesis. Treatment with drugs such as cytochalasin D (CD) or latrunculin A (Lat A) that antagonize the function of the actin cytoskeleton result in short, stout, and often split and/or multipled hairs, laterals and bristles (Geng et al., 2000Go; He and Adler, 2001Go; Tilney et al., 2000Go; Turner and Adler, 1998Go). Mutations in the crinkled (ck) gene, which encodes myosin VII, produces similar, albeit less severe, morphological defects (Ashburner et al., 1999Go; Kiehart et al., 1999Go; Turner and Adler, 1998Go). The assembly of the large actin bundles that are so prominent in bristles and laterals requires the function of two actin crosslinking proteins — the products of the singed and forked genes (Bryan et al., 1993Go; Cant et al., 1994Go; Petersen et al., 1994Go; Tilney et al., 1995Go). Mutations in either or both of these genes results in bent, twisted and shortened bristles. In addition to the prominent actin bundles developing, bristles and laterals contain a large number of centrally located microtubules. The injection of microtubule antagonists such as vinblastine (VB) and colchicine (Col) into pupae results in extremely short and fat bristles and laterals (Geng et al., 2000Go; He and Adler, 2001Go). Splitting is less frequently induced by VB or Col than by CD or Lat A.

Electron micrographic studies of developing bristles suggest that actin is added at the tip and that the actin bundles are assembled from smaller modules (Tilney et al., 1996Go). Earlier studies also argued that bristles grew by extension from the tip, as the diameter of a bristle base did not change during elongation (Lees and Waddington, 1942Go). Indeed, there are many examples in the cell biological literatures that are consistent with models where the assembly of actin at the distal tip mediates the growth of cellular extensions (Alberts et al., 1994Go; Inoue and Tilney, 1982Go; Tilney and Inoue, 1982Go). However, recent evidence has shown that actin filaments are assembled in all regions of lamellipodia in cultured cells (Watanabe and Mitchison, 2002Go) and for the possible addition of actin at all locations along the large bundles in Drosophila sensory bristle (Guild et al., 2002Go). The retrograde movement of actin filaments is also seen in lamellipodia (Watanabe and Mitchison, 2002Go). We have examined actin bundles in growing bristles by fluorescence recovery after photobleaching (FRAP) and have found that actin is added to bundles at all locations along the bristle, that actin bundles are stable and that they display retrograde movement.

The mechanism that drives bristle and lateral elongation is unclear, but the prevailing model is that actin polymerization at the tip drives elongation (Tilney et al., 1996Go; Tilney et al., 2000Go). An observation that is not predicted or easily explained is that during most of the elongation period the rate of elongation is proportional to bristle length. That is, as bristle length increases the rate of elongation increases as well (Tilney et al., 2000Go). This has been interpreted as being due to a reduced number of actin filaments being assembled more rapidly, but an alternative explanation is that elongation takes place throughout the growing shaft. As the shaft gets longer there is a greater region for growth resulting in faster growth. The published studies on bristle growth involved measurements made on fixed preparations and were by necessity average values (Lees and Waddington, 1942Go; Tilney et al., 2000Go). We have carried out time-lapse observations on growing laterals and bristles in vivo and confirmed that the rate of elongation increased as lateral or bristle length increased and determined that the growth was continuous and not pulsatile.

Mutations in a number of genes give rise to branched or split bristles and laterals, and clustered and split epidermal hairs. The phenotypes are particularly dramatic in animals mutant for either tricornered or furry (Cong et al., 2001Go; Geng et al., 2000Go). These two genes appear to be part of a conserved pathway that controls cell morphology in yeast, worms and flies (Cong et al., 2001Go; Du and Novick, 2002Go; Geng et al., 2000Go; Zellen et al., 2000Go). In recent experiments we followed the growth of arista laterals in fry mutant pupae and observed that the laterals could split at any time during their outgrowth (Cong et al., 2001Go; He and Adler, 2002bGo). What was surprising was that, once a branch-point was detected, its distance from the proximal end of the lateral increased as the lateral grew. Further, each of the lateral arms distal to a branch-point also increased in length as the lateral elongated. These observations are not easily incorporated into a growth at the tip model, but are easily explained by models that incorporate growth throughout the lateral or growth from both the proximal and distal ends. We have now extended these experiments and followed lateral growth in trc mutant laterals that were multiply branched. We observed growth in all segments of such laterals. Our observations argue strongly for growth throughout the lateral.

Bristles and laterals are extremely long thin structures. Our time-lapse observations demonstrated that, except for the earliest stages, growth is extremely polarized in the axial direction. To identify factors important for this axial versus circumferential growth we examined the morphology of bristles after treatment with actin or microtubule antagonists. We found that microtubule antagonists such as VB and Col resulted in decreased axial length and a compensatory increase in width so that the volume of the bristle was not significantly changed. These data argue that the microtubule cytoskeleton is of central importance for growth being polarized in the axial direction. The antagonism of the actin cytoskeleton resulted in decreased length but only a slight, if any, increase in bristle width. This resulted in bristles of substantially smaller volume, which argues that the actin cytoskeleton is important for growth per se, but that it has at most a modest role in ensuring the highly polarized axial nature of bristle growth. We carried out a similar analysis for several mutations that alter bristle morphology. The abnormalities associated with these mutations suggest the genes are important for growth per se but not for maintaining the highly axial nature of the growth.


    Materials and Methods
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 Materials and Methods
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 References
 
Fly culture and strains
Flies were grown on standard media. Many mutant and deficiency stocks were obtained from the Drosophila stock center at Indiana University. The UAS-{alpha}-tubulin-GFP (Grieder et al., 2000Go) stock was kindly provided by Allan Spradling (HHMI/Carnegie Institute, Baltimore, MD), the tau-GFP (Murray et al., 1998Go) stock by Andrea Brand (Wellcome/CRC Institute, Cambridge, UK) and the moesin-GFP (Edwards et al., 1997Go) stock by Dan Keihart (Duke University, Durham, NC). The neur-Gal4 (Bellaiche et al., 2001Go) stock was kindly provided by Francois Schweisguth (Ecole Normale Superieure, Paris, France). The UAS-GFP-actin transgenic flies were generated in this lab. The expression of this transgene at even fairly high levels does not interfere with bristle, lateral or hair development (X. Fei, The use of GFP fusion proteins in studying Drosophila melanogaster development. MS thesis, University of Virginia, 2001). The crinkled and Bsb alleles used were isolated in this lab.

In vivo observations
Confocal observations on growing bristles were made as follows. The pupae were attached to a microscope slide with double-sided tape, dorsal side up. The pupal case was gently removed over the head and the notum. A coverslip, coated with a small volume of Halocarbon Oil (Sigma) was placed onto the pupa with both sides supported by a piece of rubber. We primarily used machrochaetae located on the dorsal thorax in our confocal experiments. Bleaching experiments primarily used the macrochaetae located on the scutellum (Hartenstein and Posakony, 1989Go). The two posterior scutellars are folded backwards and cross each other in pupal stage. Confocal observations were made on a Nikon confocal microscope in the Keck Center for Cellular Imaging (University of Virginia, Charlottesville, VA).

Bright field observations on growing laterals were done slightly differently as follows. Aged pupae were attached ventral side up to standard microscope slides with double-sided tape or clear nail polish or to shallow well slides using super glue. Pupal cases were opened anteriorly. At various time points they were examined by microscope under bright field optics in a drop of water using a water immersion objective (40x). After observation water was removed from the slides and the pupae were then kept in a humid chamber at 25°C for further development and observations. Most pupae tolerated the treatment and reached the pharate adult stage and were morphologically normal. More recently we built a chamber using rubber spacers, double-sided tape and a coverslip as described for the confocal observations. The pupae were placed on the double-sided tape ventral side up and the pupal case opened. A small amount of 10% gelatin/10% glycerol was place on the anterior end of the pupae and between the opened pupae and the coverslip. Although the optics are not quite as good as with our earlier approaches, the chamber approach is less time consuming and can be used for automated time lapse. For these time lapse experiments we made observations at intervals ranging from 5 to 30 minutes. The lateral moving so that it was no longer parallel to the plane of focus usually ended an experiment.

Bleaching experiments
Samples were viewed using a 60x oil immersion objective. The region of tissue to be bleached was controlled by adjusting the x, y stage on the microscope during low-power imaging (10% argon laser intensity) until the desired target area was in the middle of the monitor. A small bleaching area was positioned by scanning the sample with higher magnification by increasing the zoom factor. The area was then bleached with 100% argon laser intensity. To show the bleaching-recovery, the bleached region was examined at low magnification. Images were taken with 10% argon laser intensity using time-lapse imaging or manually at various time points with the same set-up.

To measure the movement of the bleached area images were opened in Adobe PhotoShop. The rule tool was used to do all the measurement. Excel (Microsoft) was used to analyze the data. Scion Image was used to generate density profile plots in both heat shock induction and bleaching experiments.

Inhibitor experiments
Drugs were injected into pupae as described previously (Cong et al., 2001Go; He and Adler, 2001Go). For the experiments reported here the injections were carried out 26-28 hours after white pupae formation. This is several hours prior to the initiation of ocellar bristle development at 28-30 hours. Adult head cuticle was mounted in euparal, examined by bright field microscopy on a Zeiss Axioskop microscope and images obtained using a Spot camera (Diagnostic Instruments). Measurements were made using the Scion port of NIH image or the software that came with the Spot camera. Bristle volume was estimated using the assumption that the bristle is a right circular cone (v=1/3{pi}r2h) as has been done by others (Tilney et al., 2000Go). For deformed or split bristles the length and volume was sometimes estimated in segments (in some cases a segment volume was estimated assuming it was a cylinder).


    Results
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 Materials and Methods
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In pupae observations on growing laterals and bristles
As a first step we carried out in vivo observations on elongating bristles and laterals. The location of the arista at the anterior end of the pupae facilitates in vivo observations by bright field microscopy (He and Adler, 2001Go). As we have noted elsewhere, short-term observations of growing laterals showed no dramatic cytoplasmic movement (He and Adler, 2001Go). We followed laterals in pupae for 3 to more than 18 hours. The length of the laterals increased in a relatively smooth fashion and the rate of elongation continued to increase throughout the period of observation (Fig. 1A). In these experiments we only analyzed laterals that were close to being parallel to the plane of focus. Some of the `noise' in the data is probably due to slight variations in the orientation of the lateral. This was often seen and routinely happened at later stages, which invariably ended our observations on individual laterals. We also measured the width of laterals at various distances from their proximal base (Fig. 1B). At any distance from the base there was an initial period when the width increased. This occurred for a short time after the elongating tip passed that position. For the remainder of our observations lateral width remained relatively constant. These observations point out how strong the axial bias of lateral growth is. We carried out similar, albeit less extensive experiments on antennal (by bright field microscopy) and thoracic (by confocal microscopy) bristles and obtained similar results.



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Fig. 1. Graphs of lateral growth in vivo. The upper panel shows growth as a function of time for a lateral that was followed for 18 hours. The lateral extended from less than 10 µm to more than 80 µm during this time period. The lower panel shows the width of this lateral at three distances from the base of the lateral. Rectangles are for a point 5 µm from the base, triangles 15 µm from the base and ovals 35 µm from the base. This lateral was nicely oriented parallel to the plan of focus and it did not move much during the experiment allowing it to be followed for longer than most. Some of the scatter in the data is probably due to slight variations in the orientation of the lateral, which both altered its length and made it difficult to measure being slightly out of focus.

 

As elongation proceeded we noticed that there was a change in the shape of the lateral distal tip. The tip region had a short taper in small, young laterals and the taper became progressively longer as the lateral elongated (Fig. 2). Similar observations were made in our limited observations on bristles.



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Fig. 2. Lateral tip shape changes during elongation. Growing laterals of various lengths are shown. (A) 3 µm lateral; (B) 9 µm lateral; (C) 17 µm lateral; (D) 29 µm lateral; (E) 40 µm lateral; (F) the distal part of a 71 µm lateral; (G) the distal part of a 123 µm lateral. The line drawing in panel A is a tracing of the outline of the lateral in that panel. The remaining line drawings were made by uniformly stretching the line drawing from A proportional to the growth of the lateral in the panel. Note that simply stretching approximates the change in shape seen with growth. Panels D, E and F are from a time-lapse series of a single lateral as are panels B and C. Panels A and G are from the same experiment and were chosen to widen the range of lengths.

 

The early stage of lateral outgrowth involves a switch from isotropic to axial growth
To determine whether lateral growth was so strongly oriented axially at all times, we examined the earliest stages of lateral development in more depth. The first sign of lateral outgrowth is a bulging of the lateral producing cell (Fig. 3). This bulging appears isotropic in terms of the proximal distal axis of the arista. In favorable preparations the nucleus of the bulging cell is visible and it is centrally located along the proximal/ distal axis of the cell. More central core cells display this bulging than go on to produce a lateral. The next stage in lateral outgrowth entails the movement of the nucleus to the proximal side of the cell and the further bulging of the cell although now it is preferentially localized to the distal side of the cell. From this bulged region the lateral extends. This asymmetry is only seen in cells that go on to form a lateral. These observations suggest that there is a switch from isotropic or circumferential to axial growth that is associated with the commitment to produce a lateral. When the lateral first starts to elongate the tip is sometimes irregular in shape but this rather quickly becomes symmetrical. As noted previously, nuclei of all central core cells migrate proximally (He and Adler, 2001Go), so the proximal migration of the nucleus is not linked to lateral outgrowth. We have not been successful in examining the earliest stages of bristle outgrowth.



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Fig. 3. Early stages of lateral growth. Panels A-D show a time-lapse series just prior to the start of outgrowth. The lateral is shown at 25 (A), 26 (B), 27 (C) and 28 (D) hours after white pupae formation (awp). Panels E-I show a time lapse series for the early stages of lateral outgrowth. The lateral is shown at 28 (E), 29.5 (F), 31 (G), 33 (H) and 35 (I) hours awp. Panel J is a cartoon representation of the early stages in lateral outgrowth. From top to bottom of panel J are drawings representing aristae at 20, 25, 28, 31 and 35 hours awp. Note, as lateral development proceeds the cells become highly elongated along the proximal distal axis and the nuclei move proximally (He and Adler, 2001Go). The distorted shape of the 35 hour cell is meant to represent this.

 

The microtubule cytoskeleton is essential for the bias toward axial growth
The long thin morphology of a mature bristle or lateral demands that growth be highly axial and this is likely to require highly organized intracellular transport of `bristle material'. In other cell types the microtubule and/or actin cytoskeletons have been found to be important for directing intracellular transport (Goode et al., 2000Go) and previous experiments have found that disruption of either the microtubule or actin cytoskeletons results in morphologically abnormal bristles and laterals (Geng et al., 2000Go; He and Adler, 2001Go; Tilney et al., 2000Go). We reasoned that a treatment that disrupted the bias towards axial as opposed to circumferential growth would result in shorter and fatter bristles of relatively normal volume. In contrast, a treatment that simply inhibited growth would result in shorter and thinner bristles and a treatment that specifically inhibited axial growth (but which did not redirect growth) would result in shorter bristles of normal width. To assess the possible role of the cytoskeleton in both growth and the axial bias of growth we measured ocellar bristles of flies that had been injected with drugs just prior to the time of bristle initiation. We estimated the volume of the bristles by assuming that they were right circular cones, as is described in Materials and Methods. The disruption of the microtubule cytoskeleton resulted in bristles that were much shorter and fatter than normal (Table 1, Fig. 4). In the most extreme cases, all that was left of the bristle was a `blob' or `bump' of pigmented material. In our quantitative analysis of VB-treated bristles we ignored these most extreme examples. There was no significant change in bristle volume after VB treatment, which suggests that VB disrupted the axial bias of bristle growth but not overall growth. In contrast, disruption of the actin cytoskeleton resulted in bristles that were substantially shorter than normal and of reduced volume (Table 1; Fig. 4), suggesting that the actin cytoskeleton has a specific role in axial growth.


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Table 1. Ocellar bristle shape and the cytoskeleton

 


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Fig. 4. Bristle pictures. Shown are ocellar bristles from Oregon R (Ore R, wild-type, injected with saline), Ore R injected with vinblastine (VB), Ore R injected with latrunculin A (Lat-A), Sb/+ and Sb +/+ Bsb2 flies. All micrographs are at the same magnification (x360). Note the multiply split bristle that resulted from latrunculin A injection. Note the very short and fat bristles that resulted from VB injection. In the extreme VB-induced phenotype panel note that all that is left of some bristles is a pigmented `blob' (arrows).

 

Many mutations are known that result in highly abnormal bristle morphology. We carried out measurements of several of these including ck, Sb/+, Sb +/+ Bsb2 and sn f. Since ck encodes a myosin VII and sn and f actin-bundling proteins, these genotypes serve as an additional probe of the role of the function of the actin cytoskeleton. All of these mutations significantly reduced bristle length and volume (Table 1; Fig. 4). Only two of them (Sb Bsb/++ and sn f) resulted in increased bristle width and the increase was quite modest. This is particularly true given the dramatic effects of these mutations on bristle length. The analysis argues that these mutations inhibit axial growth but have little effect on circumferential growth.

Growth takes place in all regions of split laterals
Previous time-lapse observations on the elongation of mutant laterals suggested that growth could not be restricted to the distal end (Cong et al., 2001Go; He and Adler, 2002bGo). The data were compatible with growth at both the distal tip and the base or with growth throughout the lateral. To distinguish between these possibilities we examined laterals that were multiply split. Null mutations in tricornered (trc) are recessive lethals, but flies that carry the hypomorphic trc8 allele typically survive to adulthood and show a weak trc phenotype that includes one or more branches in most laterals (Geng et al., 2000Go). We followed lateral development in trc8/trc1 mutant pupae. We observed the growth of multiply split laterals on 11 separate aristae. An example of such an experiment is shown in Fig. 5. Laterals were seen to split at a wide range of developmental stages. As development proceeded the distance from the proximal base of the lateral to the proximal most branch-point increased, as did the distance between the proximal and distal branch-points. We also saw an increase in the length of the arms distal to branch-points. Thus all segments of developing branched laterals increased in length. Growth was substantial in all segments in the example shown in Fig. 5; however, the relative increase in the length of different segments was variable from lateral to lateral [e.g. see Fig. 7 in Cong et al. (Cong et al., 2001Go) for an example of where most of the increase was between the base and the branchpoint].



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Fig. 5. Analysis of multiply branched laterals argues that growth is distributed all along the proximal distal axis. The micrographs on the left are of a trc8/trc1 pupae. The developmental rate of such mutant pupae is variably delayed. The experiment started at 32 hours after white pre-pupae. The lateral is unbranched in panel A, but a branch is visible in B. A second branch can be seen in C. The graph on the right plots distance from the base of the lateral to a tip or branch point. The original tip and the furthest from the base is shown by an open circle. The second tip that resulted from the first branching is shown by an open square. The first branch point is shown by an open triangle. The tip that results from the second split is indicated by a filled circle and the second branch-point by a filled triangle. Note that the distance between all of these landmarks and the base and from one another increases over time. A cartoon is also present showing a lateral prior to branching and shortly after branching. Three models are presented for subsequent elongation. In {alpha} growth is restricted to the distal tip (represented by filled areas). This leads to no movement of the branch-points but an increase in the lengths of the arms. In ß growth is restricted to the base of the lateral. This leads to an increase in the distance from the base of the lateral to the proximal branch-point. No increase is seen in the length of the arms or of the distance between the branch-points. In {gamma} growth is distributed throughout the lateral and there is an increase in the distance of the branch points to the base, between the two branch points and in the lengths of the arms. In {Delta} growth takes place at both the base and at the distal tip. There is an increase in the distance from the base to the branch points but no increase in the distance between the branch points. There is an increase in the lengths of the arms. Our observations routinely fit the model shown in {gamma}.

 


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Fig. 7. FRAP of GFP-actin. Maximum projections of confocal stacks of scutellar bristles from a hs-GAL4/+; UAS-GFP-actin pupa are shown. Panel A shows the bristles immediately after bleaching. The other micrographs are equivalent images taken with a 1 hour interval. Panels A, C and E contain an insert showing a higher magnification view of the bleached/non-bleached boundary. Note that the distinction between the bleached and unbleached region remains obvious for the entire 5 hour experiment. The retrograde movement of the actin is also obvious. Panel G is a plot of the distance from the distal edge of the proximal unbleached region to the boundary of the socket cell (see arrow). This approach was taken as the actin bundles extend down into the cell and their proximal end was sometimes out of the range of z sections we obtained. The measurements in G are for the bristle on the right. This distance decreased over time and provides a measure of the retrograde movement of actin filaments. Note that the bleached/unbleached border becomes uneven over time due to the differential retrograde movement of individual actin bundles. Panel H is a plot of the fluorescence intensity at locations approximately 5 µm either distal or proximal to the bleached/unbleached border. Note that the proximal region of these bristles was substantially brighter than the distal region, which may be a consequence of the heat shock hours prior to the start of the experiment. There is a slight decrease in the intensity of the bright proximal unbleached region and a slight increase in the intensity of the bleached regions over time.

 

Proteins add to all regions of growing extensions
To determine whether there was any special location within a bristle, lateral or hair where newly added proteins were found we carried out a set of experiments where we induced the expression of GFP fusions with actin, {alpha}-tubulin, moesin, Dcdc42, Tricornered, tau and a membrane-tagged GFP. In these experiments we examined fixed material for all three types of extensions (bristles, laterals and hairs). We also carried out time-lapse confocal imaging of growing bristles for several of the fusions. To a first approximation GFP fluorescence appeared evenly distributed along the proximal-distal axes of the extensions. Some variation was seen that appeared to be due to curvature of the extensions and to the variation in the thickness along the extension, although we were often able to minimize these complications by examining both optical sections and projections of stacks of images obtained by confocal microscopy. A serious limitation of these experiments is the several hours that are needed for the induction of enough GFP fusion protein to be detectable. The mobility of the proteins in the cell could easily be great enough to prevent us from detecting a specific location for addition of a protein. To assess the mobility of proteins in the elongating cellular extensions we carried out fluorescence bleaching/recovery experiments on three of the GFP fusion proteins as described below.

Protein mobility in growing bristles
In one set of experiments we bleached bristles containing a membrane tagged GFP (mCD8-GFP) (Lee and Luo, 1999Go). This artificial protein has no function in Drosophila and is used as a reporter of bulk membrane protein behavior. The mobility of this marker in the membrane was great enough that we never saw a clean demarcation between the bleached and unbleached segments. Within 20 minutes we were unable to detect a bleached region. In another set of experiments we bleached bristles that were expressing a GFP-{alpha}-tubulin fusion protein (Grieder et al., 2000Go). When the bleached segment was at the tip of a bristle fluorescence recovered in about 30 minutes, whereas when the bleached segment was in the center of the bristle the recovery was somewhat slower and took almost an hour (Fig. 6). In these experiments we were initially able to detect a sharp bleached/unbleached boundary. Along with an increase in the level of fluorescence in the bleached region there was a relative decrease in fluorescence in the unbleached area near the bleach/unbleached boundary. This suggests that there is a movement of GFP-{alpha}-tubulin proteins from the unbleached to bleached region. Our experiments did not allow us to determine whether the movement involves microtubules or tubulin subunits.



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Fig. 6. FRAP of tubulin-GFP. Maximum projections of confocal stacks of a scutellar bristle from a UAS-GFP-tubulin/+; neur-GAL4/+ pupa are shown. Prior to bleaching (A) the entire bristle shows strong fluorescence. The bristle immediately after bleaching is shown in B and the recovery of fluorescence is shown in micrographs taken at 5 minute intervals. Panels B, D and H also contain an insert showing the bleach/non-bleached boundary at a higher magnification. Shown below is a graph plotting fluorescence intensity as a function of time. The intensity was measured from the maximal projections (with no other manipulation) as locations approximately 5 µm proximal or distal to either the proximal or distal bleached/unbleached border. Note the gradual increase in fluorescence in the bleached region and the gradual decline of fluorescence in the unbleached region.

 

We also carried out similar bleaching experiments on bristles that contained GFP-actin (Fig. 7). The bleached area could be discerned for 5 hours or more indicating that the large actin bundles are quite stable. This is consistent with their high degree of crosslinking (Tilney et al., 1995Go). We often saw a gradual increase in the level of fluorescence in the bleached region. We interpret this as being due to the addition of new GFP-actin protein to the existing actin bundles. We routinely detected the retrograde movement of the large actin bundles. The average rate of movement was 3.7 µm/hour for 14 bristles analyzed this way. Individual measurements varied from approximately 2-5 µm/hour. There was no significant change in the overall length of the bleached region. The border between the bleached and unbleached regions typically became irregular over time owing to a loss of register between the bleached/unbleached borders of individual actin bundles. Thus, it appears that the retrograde movement of the individual filament bundles is independent from one another. We suspect that during elongation there is depolymerization of actin bundles at their proximal end (e.g. Guild et al., 2002Go). However, our data are not compelling on this point. To minimize bleaching we did not routinely image much below the apical surface of the socket cell. Thus, in many experiments we cannot be certain of where the proximal end of the actin bundles was located and how much if any this moved during bristle growth.

In the experiments with GFP-{alpha}-tubulin we routinely noticed a cone-shaped region with a very low level of fluorescence located centrally and at the base of the shaft. This unlabeled space became less obvious in older bristles, which appeared to have a lower microtubule density at the base. On occasion a region of this cytoplasm appeared to split off from the remainder and it then migrated distally (Fig. 8). The length of the bristle and the distance of the unlabeled space to the tip of the bristle were measured to analyze the movement. The data shows that bristle was elongating during the observation period, while the distance of the unlabeled space to the tip of the bristle decreased slightly. Thus, the region of central cytoplasm was moving slightly faster than the bristle was elongating.



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Fig. 8. The movement of central cytoplasm in developing bristles. Shown is a hs-GAL4/+; UAS-GFP-tubulin/+ pupae. At the start of the experiment the distal segment of the bristle was bleached. The arrow points to the distal tip of the bristle and the arrowhead to a faintly fluorescent region in the proximal central region of the bristle. Below is a graph that plots the length of the bristle (rectangles) and the distance from the distal edge of the faintly fluorescent region to the tip of the bristle (ovals) as a function of time. The bristle grows more than 20 µm during the experiment. The faintly fluorescent region moves distally at a slightly faster rate than the tip so that the distance from the faint region to the tip decreases slowly over time.

 


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 References
 
Growth is distributed along the entire length of a bristle or lateral
Time lapse observation of lateral and bristle development showed that the rate of growth is a direct function of bristle or lateral length. This is consistent with previous observations on fixed material (Lees and Waddington, 1942Go; Tilney et al., 2000Go) and is consistent with the hypothesis that growth takes place at all locations along the proximal distal axis of the extension. Strong support for this hypothesis came from the experiments where we followed the growth of trc laterals. In these experiments we used branch points on the mutant laterals as fiduciary landmarks. We observed an increase in the distance between the proximal base of the lateral and the proximal most branch point, between adjacent branch points and between the distal most branch point and the distal tip of the lateral (Fig. 5). Unless branch-point migration occurs, these observations provide direct evidence for growth at all locations along the lateral. We cannot rule out the possibility of migration, but we have seen no evidence for it (e.g. a decrease in the distance between a branch point and the base of a lateral) in time-lapse observations of more than 50 branched laterals of various genotypes [e.g. trc, fry, sn f, mwh, in, fy, ck, koj or Bsb laterals (Cong et al., 2001Go; He and Adler, 2002aGo; He and Adler, 2002bGo)]. The mechanism that drives the elongation of bristles and laterals is unclear. We think it is unlikely to be primarily due to polymerization of actin at the tip of the growing extension, as this cannot easily explain the evidence for growth at all positions along the proximal distal axis. One possible mechanism is the sliding of microtubles both proximally and distally. EM evidence supporting this possibility was reported previously (Tilney et al., 2000Go). The sliding of the large actin bundles relative to plasma membrane (or microtubules) is also a potential source of energy for elongating laterals and bristles. This is consistent with previous observations that implicate the function of the actin cytoskeleton in bristle and lateral elongation (Cong et al., 2001Go; He and Adler, 2001Go; Tilney et al., 2000Go; Turner and Adler, 1998Go).

During elongation the shape of the distal tip of both bristles and laterals changes in similar ways. In both cases the tapered region is short in short extensions and gets gradually longer as the extension extends. This is predicted by models that incorporate growth throughout all regions of an extension (Fig. 2). Growth in the tip region will increase the distance over which the taper is seen. This observation cannot be explained by models that rely on growth only at the base. Models that assume growth at the tip requires an additional ad hoc assumption to explain the increased taper.

Axial growth of laterals and bristles
The growth of bristles and laterals must be highly biased axially to achieve their long thin shape. Our time-lapse observations argue that for much of the growth of these structures this bias is close to complete. The location of the arista at the anterior edge of the pupae has enabled us to carry out time-lapse imaging of the initial stages of lateral development. Our observations suggest that there is an initial short period of isotropic growth followed by a transition to axial growth. Inhibitor studies suggest that the microtubule cytoskeleton has an important role in the development or maintenance of the highly axial nature of bristle and lateral growth. We are not aware of any mutations that produce bristles with morphological defects equivalent to those we are able to induce by the injection of vinblastine or colchicine (Geng et al., 2000Go). Thus, genetic data supporting this hypothesis is lacking. In contrast, antagonism of the actin cytoskeleton resulted in shorter but not markedly wider bristles, suggesting that the actin cytoskeleton is important for axial growth but not for the striking axial bias. This was also caused by mutations in genes such as ck that encode proteins that interact with the actin cytoskeleton. This provides convincing genetic data that the actin cytoskeleton is functionally important for axial growth. One limitation of our experiments is that we were unable to score bristles that resulted from treatment with a high level of actin antagonists as such a treatment resulted in extensive bristle splitting and other deformities that made it difficult to make accurate measurements. It is possible that the apparent qualitative difference between actin and microtubule inhibitors would diminish if we were able to examine quantitatively the effects of high doses of actin antagonists.

The apparent transition from isotropic to axial growth that we detected in laterals brings to mind the axial to isotropic growth transition and the switch to filamentous growth that is seen in budding yeast. A number of genes have been identified as playing an important role in these processes. Among the genes identified as being essential for axial growth are Cbk1 (Bidlingmaier and Snyder, 2002Go; Colman-Lerner et al., 2001Go; Racki et al., 2000Go) and Pag1p (Du and Novick, 2002Go). Interestingly, the Drosophila homologs of these two genes are tricornered and furry. Loss of function mutations in these genes results in split and multipled laterals, and split and occasionally deformed bristles (Cong et al., 2001Go; Geng et al., 2000Go). The mutant phenotypes in the fly do not show evidence of a failure of axial growth, but it is possible that the splitting phenotype is in fact due to an abnormal organizing center for promoting axial growth. Mutations in the worm homologs of these genes result in increased neurite branching (Zellen et al., 2000Go). The basis for this could be similar to the split bristles and laterals seen in Drosophila.

Cytoplasmic movements
The cytoplasm of extending bristle shafts is notably stable when examined by bright field microscopy. To determine whether this was the case for all or specific molecular components of the bristle we used FRAP. We found the large actin bundles to be stable, consistent with their highly crosslinked nature. We detected a retrograde movement of actin bundles of on average 3 µm/hour. Retrograde movement is seen in other cellular extensions such as lamellipodia (Watanabe and Mitchison, 2002Go) and is likely to be a general feature of such extensions. Interestingly, the movement of neighboring bundles was not strictly coordinated. Although all bundles moved proximally the rate of movement varied enough so that the boundary between the bleached and unbleached segments became jagged. This observation suggests that there is no stable linkage between neighboring actin bundles. The polarity of all of the actin filaments within a bundle is consistent (Tilney et al., 1996Go), thus a population of membranelinked myosin motor protein would interact in a consistently polarized way with the actin bundles and could provide force for the retrograde movement of the bundles and the distal movement of membrane.

In the experiments where we bleached GFP-{alpha}-tubulin we found the mobility of the GFP-tagged proteins was too great to allow us to determine whether there was any bulk movement proximally or distally. At a local level it is likely that tubulin is moving both proximally and distally as the intensity of unbleached segments on both sides of bleached segments decreased slightly as the fluorescence of the bleached area recovered. We were not able to determine in these experiments whether the movement of tubulin was due to the movement of microtubules or of monomer protein. The fortuitous observation of cytoplasmic regions deficient for GFP-tubulin provided evidence for the distal movement of centrally located cytoplasm in developing bristles. Taken together, a picture emerges where cytoplasm in the center of the bristle moves distally, while peripherally located actin bundles move proximally. Presumably there is a greater mass of material that moves distally to allow for the elongation of the bristles.


    Acknowledgments
 
This research was supported by a grant from the NIGMS to P.N.A. We also acknowledge the support of the University of Virginia Cancer Center and the Keck Center for Cellular Imaging. We thank Jeannette Charlton for her help in many aspects of the project.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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