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First published online 14 December 2005
doi: 10.1242/dev.02191
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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.
* Author for correspondence (e-mail: hdbrown{at}u.washington.edu)
Accepted 31 October 2005
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, Axon remodeling, Ecdysone receptor, Metamorphosis
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). This
rewiring proceeds through programmed cell death of larval neurons, maturation
of adult-specific neurons, and remodeling of persistent larval neurons for
adult use (Tissot and Stocker,
2000; Truman,
2005; Weeks,
1999). These persistent neurons prune back their axonal and
dendritic branches early in metamorphosis and then grow new, adult-specific
arbors, establishing the neuronal circuitry necessary for the adult. The
ecdysteroids, particularly 20E, act through their receptor to mediate pruning
and outgrowth in the nervous system of Drosophila melanogaster. The
ecdysone receptor is an obligate heterodimer formed from two nuclear
receptors: EcR and the RXR-ortholog Ultraspiracle (USP)
(Koelle et al., 1991;
Yao et al., 1992). The EcR-USP
heterodimer binds to specific ecdysone response elements (EcRE) in DNA to
regulate transcription of target genes
(Cherbas et al., 1991).
The EcR/USP complex, referred to in this paper as the ecdysone receptor, is
able to both activate and repress target genes, depending on the presence or
absence of ecdysteroids (reviewed by
Kozlova and Thummel, 2000).
When ecdysteroid is absent and sufficient amounts of a co-repressor such as
SMRTER are present, the ecdysone receptor is able to repress transcription
(Dressel et al., 1999;
Tsai et al., 1999). When
ecdysteroid is present, it binds to the ecdysone receptor complex, causing a
conformational change that promotes the release of co-repressors and the
binding of co-activators, initiating transcription of early response genes
(Bai et al., 2000;
Sedkov et al., 2003). Both the
activational and repressive actions of the receptor are functionally
significant in vivo. For example, USP null clones in the wing disc show a
failure to express some ecdysone target genes, while others are expressed
prematurely, and the clones show precocious differentiation of sensory neurons
(Schubiger and Truman, 2000).
This mixed response suggests that although the steroid acts via the EcR/USP
complex, in some cases it is through activation while in others it is through
derepression.
EcR is a member of the nuclear receptor superfamily. Its N-terminus is
composed of a variable A/B domain, and may contain a ligand-independent
activational region (AF1). The E domain contains the main dimerization domain
and another activational region (AF2) contingent on ligand-binding
(Robinson-Rechavi et al.,
2003). There are three EcR isoforms in D. melanogaster:
EcR-A, EcR-B1 and EcR-B2. Studies have shown that EcR-B1 and EcR-B2 both have
strong activation functions in their A/B regions, while EcR-A may have an
inhibitory function (Hu et al.,
2003; Mouillet et al.,
2001). All three EcR isoforms bind equally well to the DNA EcRE
(Cherbas et al., 1991;
Mouillet et al., 2001) and to
ecdysteroid (Dela Cruz et al.,
2000), but they have different spatial and temporal expressions
and induce different cellular responses
(Cherbas et al., 2003;
Talbot et al., 1993;
Truman et al., 1994).
Variable cellular responses to the three EcR isoforms are particularly
evident in the remodeling nervous system. High levels of EcR-B1 early in
metamorphosis have been associated with pruning of larval branches
(Truman et al., 1994). In many
remodeling neurons, EcR-B1 expression decreases and EcR-A expression becomes
prominent as the neuron begins its adult outgrowth. By contrast to remodeling
larval neurons, arrested imaginal neurons born during larval life express only
EcR-A at the start of metamorphosis as they begin their adult outgrowth
(Truman et al., 1994).
Experiments examining pruning in both thoracic ventral (Tv) neuron dendrites
(Schubiger et al., 1998) and
mushroom body axons (Lee et al.,
2000) showed that EcR-B mutants lose their ability to prune, and
can be rescued by expression of either EcR-B1 or EcR-B2 but not EcR-A
(Lee et al., 2000;
Schubiger et al., 2003;
Schubiger et al., 1998).
EcR-B1-specific mutants prune normally, indicating either that B1 and B2 are
functionally redundant or that B2 is the primary isoform driving pruning
(Schubiger et al., 1998).
These experiments suggest that the EcR-B isoforms are associated with pruning
and reorganizational responses while EcR-A is responsible for outgrowth of
arbors. However, the specific role each EcR isoform has in directing precise
cellular responses during neuronal remodeling is still not understood.
In this study, we investigated the role of EcR in Tv cell axonal remodeling. Our results highlight the specific axonal events during Tv cell remodeling, allowing us to analyze in detail both pruning and outgrowth. Results from cell-autonomous expression of EcR dominant negative constructs and EcR RNAi show that, while activation is necessary for early pruning events, it may not be required for later remodeling events.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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) and Cherbas
et al. (Cherbas et al., 2003).
Similar constructs based on the A and B2 isoforms of EcR,
UAS-EcR-AF645A, UAS-EcR-AW650A and
UAS-EcR-B2W650A were made as follows: the 5' portion of the
EcR-B1 coding sequence was excised from a mutant CMA-EcR-B1 plasmid with
BamHI (which cuts upstream of the start of translation) and
AscI (which cuts within the coding sequence for the common-region
DNA-binding domain), and replaced with the corresponding
BamHI-AscI fragment from either CMA-EcR-A or CMA-EcR-B2
(Hu et al., 2003) to generate
a coding sequence containing the A or B2 isoform-specific N-terminus and the
dominant negative mutation (F645A or W650A). The entire coding region for the
mutant EcR was then excised with BamHI and NheI and inserted
between the BglII and XbaI sites of the vector pUAST, to
generate the responder transposon. UAS-EcR-B1F645A,
UAS-EcR-B1W650A, UAS-EcR-B2W650A and
UAS-EcR-AW650A were inserted into flies via P-element
transformation to create homozygous fly stocks
(Cherbas et al., 2003). EcR
RNAi was performed using the CA104 stock containing an inserted EcR core
inverted repeat construct (UAS-IR-EcR) from C. Antoniewski
(Schubiger et al., 2005). To
facilitate cell-autonomous expression of these constructs in the Tv cells, the
above stocks were crossed with a fly stock containing both the GAL4 driver
(Brand and Perrimon, 1993)
under control of the FMRF-amide promoter and a UAS-GFP insert
(Lee and Luo, 1999) that
targets CD8::GFP to the membrane. The ywUASmCD8::GFP; FG10 stock made use of a
FMRF-amide promoter construct created by S. Robinow, University of Hawaii
(Schubiger et al., 2003;
Suster et al., 2003). Progeny
expressed both membrane-bound GFP and either EcR dominant negative construct
or IR-EcR (core) cell autonomously in the Tv neurosecretory cells.
Immunocytochemistry
Central nervous systems were dissected and fixed for 20-30 minutes in 4%
paraformaldehyde, followed by multiple rinses with PBS containing 1% Triton
X-100 (PBS-Tx). Tissue was blocked with 5% normal donkey serum for 15 minutes
followed by transfer to a solution of primary antibodies and incubation
overnight. Primary antibodies used were monoclonal mouse anti-SCP
(Masinovsky et al., 1988), rat
anti-mCD8 (1:200, CALTAG) and mouse anti-EcR (IID9.6, common)
(Talbot et al., 1993). After
several rinses with PBS-Tx, the tissues were incubated overnight in secondary
antibody (1:200 in PBS-Tx of Texas red conjugated donkey anti-mouse IgG, Texas
red conjugated donkey anti-rabbit IgG, or FITC conjugated donkey anti-rat IgG;
Jackson ImmunoResearch). Nervous systems were rinsed several times in PBS and
attached to polylysine-coated coverslips, then dehydrated, cleared in xylene
and mounted in DPX (Fluka). For quantification of EcR or mCD8
immunoreactivity, all genotypes to be compared were dissected, antibody
stained and imaged as one batch.
Staging and imaging
Animals were collected at white puparia and maintained at either 25°C
or 29°C. Late wandering larvae were distinguished by their enlarged
salivary glands. For live imaging, whole intact nervous systems were dissected
from staged animals, leaving the ring gland intact. Tissue was attached to a
polylysine-coated coverslip and inverted onto a metal imaging chamber
(Kiehart et al., 1994). This
chamber was bounded on the bottom by an oxygen-permeable membrane (model 5793,
YSI, Yellow Springs, OH) and filled with Shields and Sang M3 insect culture
media (Sigma, St Louis, MO) with 7.5% fetal bovine serum (Sigma). For live
imaging, a Bio-Rad Radiance 2000 confocal microscope equipped with a 488 nm
Kr/Ar laser was used. Individual Z-stacks with a step size of 1.05
µm were taken every 20 minutes over a 3-18-hour period. The development of
explanted nervous systems slowed in culture, and 2 hours of time in vitro was
roughly equivalent to 1 hour in vivo
(Gibbs and Truman, 1998). The
age of Z-stack projections from time-lapse video was calculated by
referring to the time of explantation. Time-lapse movies were created from the
Z-stacks using NIH ImageJ
(http://rsb.info.nih.gov/ij/).
Analysis
Arbor area, arbor footprint, filopodial quantification and quantification
of EcR and mCD8 immunoreactivity were calculated using NIH ImageJ. For arbor
area, all images were adjusted to a similar threshold, converted to black and
white, and quantified by counting the number of pixels. To obtain the arbor
footprint, a polygon was created by connecting the outer edges of the axon
arbor with straight lines; then the total number of pixels in the area inside
the polygon were counted. Filopodia were quantified by manual counting at each
hour for each neuromere. For more developed arbors that were already
fasciculated, all filopodia were counted and divided by three to get a
filopodia count for each neuromere. To quantify relative EcR and mCD8
immunoreactivity, fixed cell bodies were imaged from each genotype. Each
nucleus was selected and the mean intensity determined using ImageJ. The mean
intensity for the background area was subtracted from the selected area to get
the net mean intensity for each nucleus. As all animals used had one copy of
mCD8::GFP, the immunoreactivity of mCD8 should be similar among all genotypes.
The ratio of EcR to mCD8 immunoreactivity was calculated by dividing the net
mean EcR intensity by the net mean mCD8 intensity. To calculate statistical
significance, a non-paired t-test (
=0.05) was run using
SigmaStat and SPSS software.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The neurohemal organs project above the dorsal surface of
the central nervous system (CNS), while the somata and dendrites of the Tv
cells reside within the neuromere (Fig.
1A). Although the central dendrites of these cells had severely
pruned back by 5-6 hours after puparium formation (apf)
(Schubiger et al., 1998), the
start of axonal remodeling did not begin until approximately 10 hours apf,
with the larval arbor being completely pruned by 18 hours apf. Outgrowth of
adult branches began between 18 and 20 hours apf, and was essentially finished
by 48 hours apf, forming a complex mesh-like arbor spreading out over the
dorsal surface of the CNS (Fig.
1C). The branching of the arbor changed little between 48 hours
and adult eclosion (approximately 96 hours apf).
We used confocal live imaging to visualize axon pruning and growth in
short-term explanted Drosophila nervous systems. The Tv cells were
vitally labeled using the FG10-GAL4 driver to express the membrane marker
CD8::GFP in these cells (Fig.
1B). The superficial location of the Tv cell axons on the dorsal
surface of the explanted CNS aided live imaging. At pupariation, the
neurohemal sites appeared much the same as in the larva and we observed no
morphological changes from late third instar through 3 to 5 hours apf.
Beginning around 5 hours apf, however, fine filopodia started sprouting from
the axon arbor (Fig. 2A), and
approximately 5-7 hours later the neurons began pruning back their axonal
arbor. During pruning, we saw active filopodia both on the regressing larval
portion of the arbor and in an adult growth zone that was forming beneath the
neurohemal organ (Fig. 2B).
Most filopodia present on the neurohemal organ and the growth zone changed
considerably in length during each 20-minute interval, either extending,
shortening or branching. In instances in which we could follow the regression
of individual axon branches, we saw only retraction and no sign of branch
fragmentation (local degeneration) as described for mushroom body axons
(Watts et al., 2003) and
dendrites of the dendritic arborizing neurons
(Williams and Truman, 2005).
Addition of hemolymph to the culture, supplying a source of hemocytes, did not
affect pruning, and no severing of branches occurred (data not shown).
Filopodia in the growth zone area continued to be active as the larval axonal
arbor disappeared, and eventually stabilized to form branches at the base of
the pruned neurohemal organ (Fig.
2B-C). We observed filopodia on both the shaft and tip of primary
branches as they extended to form a mesh-like network of axonal branches.
Filopodia persisted as late as 56 hours apf, as the arbor continued to expand
through activity on the outermost branches of the network (not shown).
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). The F645A mutation is in the co-activator binding
groove in the AF2 domain and results in a receptor that binds ligand but
cannot mediate activation. The W650A mutation prevents steroid binding and,
consequently, also results in a lack of ligand-dependent activation. In vitro,
these EcR-DNs bind DNA and USP normally, and act as competitive inhibitors of
endogenous wild-type EcR (Cherbas et al.,
2003). As both types of EcR-DNs are equally effective at
suppressing ligand-dependent activation, differences between the biological
effects of the two are probably due to the fact that one can bind steroid and
the other cannot. The latter ability is significant, because the unliganded
EcR/USP complex can act as a repressor. Both the F645A and W650A-based EcR-DNs
are capable of mediating this repression
(Hu et al., 2003), but their
differences in ligand binding may mean that one is a conditional repressor
whereas the other is a constitutive repressor (see Discussion).
We compared the effects of the F645A or W650A substitution in EcR-B1 and
the W650A substitution in all three isoforms (EcR-B1, EcR-B2 and EcR-A).
Because endogenous EcR shows no isoform specificity in binding to DNA
(Mouillet et al., 2001), we
assumed that expressing EcR dominant negative at high levels in the cell
displaced the endogenous EcR in a non-isoform-specific manner. The different
A/B regions of the EcR-DNs should have little effect on the activation
capacities of the modified receptor, because the AF2-mediated activation
functions are blocked in all of them. However, since the EcR-DNs retain their
repressor function, the use of isoform-specific W650A-based EcR-DNs allowed us
to probe the role of the A/B region of the isoforms in processes that might be
mediated via derepression. Comparison of the F645A mutation to the W650A
mutation in EcR-B1 let us analyze the need for activation over derepression of
the AF2 domain.
Cells expressing the individual EcR-DN constructs showed similar pruning responses, regardless of which construct was present. Fig. 3 shows the progression of pruning for neurons expressing each of the EcR-DN constructs. All cells expressing the EcR-DN were of normal size and larval morphology at the start of metamorphosis. By 18 hours apf, however, the cells expressing EcR-DN showed only modest pruning compared with control cells (expressing GFP only), and by 24 hours apf they still had a reduced larval neurohemal organ with no sign of branch outgrowth. Neurons expressing CD8::GFP only (control cells), by contrast, had completely pruned their neurohemal organs and were beginning branch outgrowth by this time (Fig. 3). Live imaging through this pruning period also gave similar results for all EcR-DN constructs. Notably, neurons expressing any EcR-DN construct lacked filopodia before and during the pruning time period (Fig. 4A). As with the immunocytochemistry, live imaging revealed slow and incomplete pruning of the larval neurohemal organ. These larval arbors were retained well past the time of pruning into the period when the cell should switch to outgrowth (Fig. 4B-C).
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Effects of dominant negative EcR expression are qualitative
The differences seen during outgrowth of neurons expressing the various
EcR-DNs could represent a qualitative difference reflecting the molecular
action of EcR or a simple quantitative effect due to varying expression levels
of the EcR-DN inserts. We addressed this issue in three different ways:
expression of the same EcR-DN constructs from different insertion sites;
comparison of neurons from animals raised at 25°C versus 29°C; and
quantitative immunocytochemistry to determine the relative amounts of protein
expressed in Tv cell bodies for all EcR-DN constructs.
We examined the arbors of cells expressing two different inserts of EcR-B2W650A and of EcR-AW650A. There was no significant difference seen between the arbor areas for the individual inserts of the same construct. This was also true for the arbor footprints; the cells expressing either of the EcR-B2W650A inserts were similar, and cells expressing either of the EcR-AW650A inserts were similar (Table 1).
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).
Therefore, we compared the arbor areas and footprints of the adult axonal
arbors for cells expressing the various EcR-DNs at 25°C versus 29°C.
The only significant difference seen was between the arbor footprints of cells
expressing the EcR-B1W650A at the different temperatures. At
25°C, the footprint pixel count was 38,578 (n=8), while at
29°C it was reduced to 12,422 (n=10, P=0.01). The
difference in the arbor area of cells expressing EcR-B1W650A at
25°C versus 29°C was not significant, with pixel counts of 3306 and
3766, respectively (P=0.41). All of the other EcR-DNs were not
significantly different for either arbor area or arbor footprint at the
different temperatures (Table
1). We also used quantitative immunocytochemistry to determine the relative protein levels in nuclei of cells expressing each individual EcR-DN. EcR levels were significantly higher in cells expressing EcR-DN than in control cells (P<0.05; Fig. 7A). Although EcR levels varied between the cells expressing different EcR-DNs, the differences in protein level did not correspond with the severity of axonal phenotype. Neurons expressing EcR-B1F645A had a relatively higher EcR level than those expressing EcR-B1W650A, but the cells expressing EcR-B1W650A had a more severe phenotype. CD8 levels in all cells examined did not differ significantly; the ratio of EcR to CD8 immunofluorescence confirmed that the higher levels of EcR in cells expressing EcR-DN was not due to differences in imaging (Fig. 7A, inset). All of the above data support our conclusion that the qualitative differences we see for the different EcR-DNs are not based on quantitative differences in the level of EcR expression.
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). We selectively expressed UAS-IR-EcR (core) and UAS-CD8::GFP
in the Tv cells, using the FG10-GAL4 driver. We quantified total EcR protein
levels as described above. In the late third instar, Tv cell nuclei from
IR-EcR (core) expressing animals showed total EcR levels below the level of
detection compared with EcR levels in control animals
(Fig. 7B). The ratio of EcR to
CD8 protein was much lower for cells containing IR-EcR (core) than those
without (Fig. 7C, inset),
indicating that the decrease in EcR immunoreactivity was due to a true
suppression of EcR protein and not due to immunocytochemistry or imaging
artifact. To further insure adequate levels of IR-EcR (core) to knock down EcR
level, we raised animals at 29°C until the time of dissection. We
compensated for difference in developmental time when comparing IR-EcR (core)
data to those from animals maintained at 25°C
(Powsner, 1935). Axonal pruning in the cells expressing IR-EcR (core) resembled the reduced pruning seen in cells expressing the dominant negative EcR constructs. Immunostained nervous systems showed normal larval neurohemal organs at pupariation, but by 24 hours apf, the cells still had larval-like arbors, although they were clearly reduced in size compared with larval stages. We did not see adult-like branches at 24 hours apf (Fig. 8A), but by 48 hours apf and in the adult a new arbor was present that appeared similar to EcR-B1F645A- and EcR-B2W650A-expressing cells. The neurons expressing IR-EcR (core) also had a dense, clumped axon arbor that was surrounded by the reduced adult-like arbor. Comparison of the arbor footprints of cells expressing IR-EcR (core) to control cell arbor footprints emphasizes the decreased branching in these cells (Fig. 8B).
Data from live imaging of cells expressing IR-EcR (core) showed no filopodia during the normal pruning phase (12-18 hours apf) along with a slowed retraction of the larval branches. We first observed filopodia around 24 to 28 hours apf (Fig. 8C). These filopodia formed stable, fasciculated branches, resulting in an adult arbor with both larval and adult characteristics, similar in appearance to arbors of cells expressing EcR-B1F645A and EcR-B2W650A. Fig. 8D shows quantification of filopodial activity of cells expressing IR-EcR (core).
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DISCUSSION |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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) or a
combination of fragmentation and retraction
(Williams and Truman, 2005).
In both of these cases, surrounding cells participate in the fragmentation
process. For the axons of the mushroom body gamma neurons, glial invasion of
the axon lobes is necessary for proper pruning
(Awasaki and Ito, 2004),
whereas for the peripheral dendritic arborizing neurons, phagocytic blood
cells are seen to sever proximal and distal regions of the dendritic arbor
(Williams and Truman, 2005).
In both immunocytochemistry of fixed preparations and time-lapse movies of
pruning in the Tv neurons, we saw no sign of fragmentation, even with the
addition of hemolymph. In instances where we could visualize individual
branches, they were gradually retracted. The fact that Tv axon pruning
occurred via retraction only may be partially due to their peripheral location
underneath the basal lamina (Lundquist and
Nassel, 1990). This location may isolate them from invading glia
within the CNS and phagocytic blood cells circulating in the hemolymph.
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). The
axonal structure, however, appears normal at this time, so it is unlikely that
these filopodia result from a disruption of the axonal cytoskeleton. The
difference in timing between dendrite and axon retraction is also seen in
motoneurons MN1-4, which innervate the larval mesothoracic muscles of the
larva and remodel to innervate the dorsolongitudinal indirect flight muscles
of the adult fly. The dendrites of these neurons undergo retraction between 2
and 4 hours apf, while the axons begin to prune at 6 hours apf and finish at
10 hours apf (Consoulas et al.,
2002). In the Tv neurons, filopodia continue to be present as the
axonal arbor is retracted. The presence of filopodia during pruning was also
observed in the dendritic arborizing neurons
(Williams and Truman, 2005).
In the mammalian hippocampus, filopodia are associated with both formation and
reduction of dendritic spines. Synaptic activation results in growth of
dendritic filopodia (Maletic-Savatic et
al., 1999), while deafferentation causes filopodial activity
followed by reduction of spine density
(Benshalom, 1989). It is
probable that the presence of filopodia is either a reflection of cytoskeletal
instability or plays an active role in disrupting or transporting the
cytoskeleton of axons being pruned by retraction.
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;
Schaefer et al., 2002). It is
unknown if these later filopodia represent a different pharmacological or
molecular class from the structures seen during pruning.
Role of ecdysone signaling in pruning
The expression of the EcR dominant negative constructs and the IR-EcR
(core) construct in the Tv neurons resulted in incomplete pruning of the
larval axonal arbors. Cells expressing any one of these constructs showed a
reduction in neurohemal organ size during the pruning period, but substantial
larval material was retained into the outgrowth phase. In a study by Williams
and Truman (Williams and Truman,
2005) examining pruning in the dendritic arborizing neurons,
proximal destabilization of microtubules and thinning of the dendrites was
seen before fragmentation. When dominant negative EcR was expressed, the
dendrites did not thin proximally. Shortening dendrites with retraction bulbs
at their tips were observed, however, indicating that some aspects of pruning
still persisted in cells whose steroid-response system was blocked. This
indicates that proximal cytoskeletal destabilization may depend on ecdysone
signaling in the da neurons, while distal tip retraction may not
(Williams and Truman, 2005).
One possibility is that distal retraction results from 20E-evoked changes in
the epidermis on which the neuron resides. Although fragmentation of Tv cell
axons does not occur, the pruning process in these cells may nevertheless be
similar to that seen in the da neurons. Destabilization of the cytoskeleton in
proximal regions of the axon arbor may facilitate rapid axon retraction.
Without activation via ecdysteroid (cells expressing dominant negative EcR or
EcR RNAi), cytoskeletal destabilization along the axon shaft may not occur and
any pruning must take place via tip retraction with slow retrograde
progression. Additionally, cells expressing EcR-DN or IR-EcR (core) lacked
filopodia during the pre-pruning and pruning stages. As filopodia are
associated with cytoskeletal instability
(Benshalom, 1989;
Maletic-Savatic et al., 1999),
this lack of filopodia may indicate that the cytoskeleton is not destabilizing
properly, thereby slowing retraction. Because pruning and early filopodial
activity were deficient in cells expressing either the EcR-DNs or IR-EcR
(core), we conclude that these events must be mediated by the ecdysteroid
activation of transcription (Fig.
9). The fact that the Tv cells prune at all suggests that either
the EcR-DNs and the EcR-RNAi are not inactivating the endogenous EcR
sufficiently, or that there may also be a non-cell-autonomous component to
pruning. As the EcR-DNs are not expressed in the supporting glial cells of the
neurohemal organ or in surrounding tissue, these support cells are still
responsive to ecdysone. The modified pruning response we see could be due to
ecdysone-mediated death of the supporting glial cells or signaling from the
surrounding tissue, causing the Tv axons to retract, albeit slowly, as
discussed above.
Earlier studies on dendritic pruning of the Tv cells and axonal pruning in the mushroom body showed that the EcR-B isoforms are necessary to support pruning, indicating the need for AF1 activation in this process. That all of the dominant negative EcR isoforms as well as the IR-EcR (core) construct also fail to prune effectively argues that activation through the AF2 domain of EcR is also essential for this process. The EcR-B AF1 and AF2 activation domains probably work cooperatively to bring about the rapid deconstruction of the axonal arbor.
Role of ecdysone signaling in outgrowth
Unlike pruning, outgrowth of Tv cell axons depends on the EcR construct
being expressed. Cells expressing EcR-B1F645A,
EcR-B2W650A or IR-EcR (core) started to extend filopodia during the
outgrowth phase, which began after 24 hours apf. This outgrowth occurred both
from filopodia that formed on the remaining larval arbor and from the growth
zone underneath. However, new branches that extended from both the larval
arbor and the neural outgrowth growth zone were unusual in their morphology.
Compared with controls, branches from both sites had small varicosities along
their length, resulting in a `blebby' appearance. In a study by Jacobs and
Stevens on cultured PC12 cells, neurites with microtubules depolymerized by
Nocodazole showed formation of varicose expansions filled with randomly
oriented membranous organelles, by contrast to untreated neurites, in which
organelles are uniformly distributed and longitudinally oriented
(Jacobs and Stevens, 1986).
The Tv neurons in this treatment group may have suffered similar microtubule
abnormalities, resulting in formation of similar irregular varicosities.
Cells expressing EcR-B1W650A or EcR-AW650A showed a
qualitatively different type of outgrowth, forming very few filopodia and
finishing with adult arbors that appeared larval-like and did not form a
net-like structure over the surface of the nervous system. This draws
attention to the importance of filopodia in guiding proper outgrowth to form
the adult arbor. Although we observed the extension of rare branches, these
seldom sub-branched and did not fasciculate with those from other Tv cells to
form the adult-like meshwork. Filopodia actin bundles guide microtubule
polymerization in Aplysia
(Schaefer et al., 2002), while
inhibition of F-actin in cultured hamster neurons leads to inhibition of
directed growth (Dent and Kalil,
2001; Schaefer et al.,
2002). Eradication of filopodia through cytochalasin treatment of
grasshopper pioneer neuron growth cones results in disoriented and improper
branch formation, but does not eliminate axon extension
(Bentley and Toroian-Raymond,
1986). The latter result is similar to our results with the Tv
cells, in that growth was not eliminated by lack of filopodia, but instead
resulted in a more larval-like arbor in the adult.
EcR dominant negatives, IR-EcR (core) and remodeling
All the constructs produced similar results for pruning, but they had
varied effects on outgrowth. These differences in the cellular response to the
various EcR constructs indicate that the molecular action of EcR on these
neurons may change during the course of their remodeling. The ability of cells
expressing IR-EcR (core) to produce filopodia and adult-like branches during
the outgrowth period is surprising in the context of 20E acting via activation
through the ecdysone receptor. It is possible that the hypomorphic levels of
EcR are nevertheless sufficient to support this activation and induce
outgrowth. However, similar types of outgrowth were seen in cells expressing
EcR-B1F645A, and activation is strongly suppressed by this dominant
negative receptor (Cherbas et al.,
2003). A more likely hypothesis is that during the outgrowth
phase, the ecdysone receptor acts predominantly as a repressor and the role of
20E is to relieve this repression (Fig.
9). The function of the ecdysone receptor as a developmental
repressor has been best studied in the developing wing imaginal disc. USP-null
clones, which cannot respond to 20E, show precocious sensory neuron
differentiation that is no longer dependent on ecdysteroid
(Schubiger and Truman, 2000).
Expression of IR-EcR (core) in the wing disc similarly leads to precocious
neuron development (Schubiger et al.,
2005). It is notable that expression of EcR-B1F645A
produces a phenotype in the Tv cells that is similar to receptor removal. As
this dominant negative can bind 20E
(Cherbas et al., 2003), our
results suggest that ligand binding by this EcR-DN may relieve the
transcriptional repression that it enforces on its target genes, thereby
enabling a core program of axonal outgrowth that does not depend on
activation. The ability of the EcR-B1F645A dominant negative to
support derepression, however, may be highly context-dependent, as there were
no differences seen in activation or inhibition in Kc167 cells expressing
either EcR-B1F645A or EcR-B1W650A
(Hu et al., 2003).
This hypothesis, that the ecdysone receptor plays a permissive role as a
repressor during the phase of neuronal outgrowth during remodeling, is
reinforced by the results from cells expressing EcR-AW650A and
EcR-B1W650A, because these cells show a more severe phenotype than
that seen in cells with knocked-down EcR levels (Figs
5 and
8). However, the phenotype of
cells expressing EcR-B2W650A was less severe in both
immunocytochemistry and live imaging than those expressing
EcR-AW650A and EcR-B1W650A. Experiments testing the
activation potential of each of the wild-type EcR isoforms indicated the
presence of a repression domain in the A/B region of EcR-A
(Mouillet et al., 2001). Only
an activation domain (AF1) has been ascribed to the A/B region of EcR-B1
(Hu et al., 2003;
Mouillet et al., 2001), but
this region is relatively long and incompletely characterized. We suggest that
the A/B region of EcR-B1 may also possess a repression domain, although this
needs to be confirmed biochemically. If this were the case, then either of
these isoforms would then be able to tether a strong co-repressor complex that
could not be removed without ecdysteroid binding. Under this hypothesis, the
weak phenotype seen with expression of the EcR-B2W650A construct
may be a function of its short A/B region. This isoform has a strong AF1
activational function in its 17 amino acid A/B region, but it is unlikely that
this short region also contains a repressive domain. Because of this, the
EcR-B2 isoform may not be able to assemble the strong co-repressor complexes
that are seen with the other isoforms. Hence, the EcR-B2 isoform may be able
to mediate strong activation but be a very poor repressor. This model would
account for the fact that in axon outgrowth, a situation where the crucial
step is the relief of transcriptional repression by ecdysteroid binding to its
receptor, cells expressing EcR-AW650A or EcR-B1W650A
have a much more severe phenotype than cells expressing
EcR-B2W650A, EcR-B1F645A or IR-EcR (core).
These studies indicate that EcR function is complex, with expression of isoforms and co-factors, hormone titer, and derepression versus activation of target genes varying through space and time. However, studies of the effects of EcR on neuronal remodeling in combination with new technology are yielding invaluable information on the role of nuclear receptors in the development of the nervous system.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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