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
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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Summary |
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Key words: Cytoskeleton, Drosophila, Bristles, Outgrowth
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Introduction |
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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., 2000;
He and Adler, 2001
;
Tilney et al., 2000
;
Turner and Adler, 1998
).
Mutations in the crinkled (ck) gene, which encodes myosin
VII, produces similar, albeit less severe, morphological defects
(Ashburner et al., 1999
;
Kiehart et al., 1999
;
Turner and Adler, 1998
). 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., 1993
;
Cant et al., 1994
;
Petersen et al., 1994
;
Tilney et al., 1995
).
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.,
2000
; He and Adler,
2001
). 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., 1996). 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, 1942
).
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., 1994
; Inoue and
Tilney, 1982
; Tilney and
Inoue, 1982
). However, recent evidence has shown that actin
filaments are assembled in all regions of lamellipodia in cultured cells
(Watanabe and Mitchison, 2002
)
and for the possible addition of actin at all locations along the large
bundles in Drosophila sensory bristle
(Guild et al., 2002
). The
retrograde movement of actin filaments is also seen in lamellipodia
(Watanabe and Mitchison,
2002
). 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., 1996;
Tilney et al., 2000
). 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., 2000
).
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, 1942
; Tilney et
al., 2000
). 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.,
2001; Geng et al.,
2000
). These two genes appear to be part of a conserved pathway
that controls cell morphology in yeast, worms and flies
(Cong et al., 2001
;
Du and Novick, 2002
;
Geng et al., 2000
;
Zellen et al., 2000
). 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.,
2001
; He and Adler,
2002b
). 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.
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Materials and Methods |
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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,
1989). 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., 2001;
He and Adler, 2001
). 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
r2h) as has been done by
others (Tilney et al., 2000
).
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).
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Results |
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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.
|
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, 2001), 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.
|
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.,
2000) and previous experiments have found that disruption of
either the microtubule or actin cytoskeletons results in morphologically
abnormal bristles and laterals (Geng et
al., 2000
; He and Adler,
2001
; Tilney et al.,
2000
). 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.
|
|
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., 2001;
He and Adler, 2002b
). 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., 2000
). 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., 2001
) for an
example of where most of the increase was between the base and the
branchpoint].
|
|
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,
-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,
1999). 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-
-tubulin
fusion protein (Grieder et al.,
2000
). 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-
-tubulin proteins from the unbleached to bleached region. Our
experiments did not allow us to determine whether the movement involves
microtubules or tubulin subunits.
|
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.,
1995). 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., 2002
).
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--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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Discussion |
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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.,
2000). 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, 2002;
Colman-Lerner et al., 2001
;
Racki et al., 2000
) and Pag1p
(Du and Novick, 2002
).
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.,
2001
; Geng et al.,
2000
). 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.,
2000
). 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, 2002)
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.,
1996
), 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--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.
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Acknowledgments |
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