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First published online October 12, 2007
doi: 10.1242/10.1242/dev.02882
Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801, USA.
Author for correspondence (e-mail:
akira.chiba{at}miami.edu)
Accepted 4 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dendrite, Slit, Robo
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Westerfield et al., 1986). As
a result, when dendrites first appear in the embryonic CNS, axons have already
laid out basic fascicle organizations. In many animals, a neuropil, the region
enriched with synapses, develops where longitudinal and commissural axon
fascicles intersect. Both the molecular heterogeneity among neuronal processes
at the neuropil and the fact that each neuron has multiple compartments
complicate the dissection of molecular mechanisms that control the development
of dendrites in the CNS.
Slit, an evolutionarily conserved signaling molecule, is expressed by
midline cells (Rothberg et al.,
1988; Rothberg et al.,
1990; Yuan et al.,
1999; Zou et al.,
2000). Slit is a large (190 kDa) secreted protein with four
leucine-rich repeats, seven or more EGF (epidermal
growth factor) domains, and a cysteine knot
(Rothberg et al., 1990). The
second leucine-rich repeat is required to bind Robo, a transmembrane receptor
expressed by neurons (Howitt et al.,
2004). Acting through Robo family receptors
(Brose et al., 1999;
Li et al., 1999), Slit repels
axonal growth cones from the midline during early neural development
(Brose et al., 1999;
Erskine et al., 2000;
Hao et al., 2001;
Kidd et al., 1999). However,
Slit expression continues through later stages of development
(Godenschwege et al., 2002;
Johnson et al., 2004). During
its emergence, the neuropil is directly contacted by Slit-bearing filopodia
from midline cells (Vasenkova et al.,
2006), and Slit becomes enriched in the neuropils
(Johnson et al., 2004). Study
in cultured mammalian cortical and sensory neurons demonstrates that Slit
signaling can promote the branching of neurites
(Ozdinler and Erzurumlu, 2002;
Wang et al., 1999;
Ward et al., 2005;
Whitford et al., 2002).
Similarly, overexpression of Slit causes abnormal axonal branching of Mauthner
neurons in zebrafish embryos (Yeo et al.,
2001). Correlation between slit gene expression and
axonal arborization has been noted in vivo, as well
(Miyashita et al., 2004;
Ozdinler and Erzurumlu, 2002;
Ward et al., 2005;
Whitford et al., 2002).
However, the distribution of Slit protein within the developing CNS has not
been described. In particular, whether or not Slit is present prior to the
onset of dendrogenesis has not been established. Also, analyses on
slit/slit mutants have so far focused on early stages of
embryogenesis and, therefore, the potential ability of Slit to control
dendritic development remains untested in vivo.
The Robo receptor has five extracellular immunoglobulin domains, three
extracellular fibronectin type-3 domains, a single transmembrane domain, and
several conserved cytoplasmic motifs (Kidd
et al., 1998). The first and second immunoglobulin domains are
required for binding to Slit (Liu et al.,
2004). In addition to growth cone repulsion
(Brose et al., 1999;
Erskine et al., 2000;
Hao et al., 2001;
Kidd et al., 1999), Robo has
also been implicated in Slit-induced branching and arborization
(Miyashita et al., 2004;
Ozdinler and Erzurumlu, 2002;
Whitford et al., 2002).
Whitford et al. (Whitford et al.,
2002) overexpressed a cytoplasmically truncated form of Robo in
cultured mouse cortical neurons leading to the inhibition of collateral
branching of neurites. This result has been interpreted as evidence for
endogenous Robo function being required during the development of dendritic
and/or axonal arbors. Like its ligand Slit, in vivo Robo expression persists
throughout neuronal differentiation. In Drosophila, Robo, as well as
three other known receptors for Slit (Robo2, Robo3 and Syndecan) are enriched
where the neuropil emerges along the longitudinal fascicles and remains
abundant there through the end of embryogenesis
(Johnson et al., 2004;
Rajagopalan et al., 2000;
Simpson et al., 2000;
Steigemann et al., 2004). Such
an expression pattern of Robo in the embryonic CNS is consistent with its
proposed role in controlling dendrogenesis. However, genetic analysis of the
role of Robo in late-stage embryos is complicated by the fact that this
receptor is also required to guide axonal and dendritic growth cones of many
neurons at earlier stages of development
(Furrer et al., 2003;
Kidd et al., 1998;
Wolf and Chiba, 2000).
In this study, we examine the role of Slit and Robo in the context of
dendritic development through in vivo single-cell analysis. We focus on the
aCC (anterior corner cell) motoneuron, one of
the first CNS neurons to generate dendrites in Drosophila embryos and
one that can be visualized and manipulated genetically at the single-cell
level (Furrer et al., 2003;
Landgraf et al., 1997). We
conducted a series of experiments using loss-of-function mutant analysis,
single-cell genetic rescue and RNAi, quantitative immunocytochemistry and
deliberate overexpression. Based on these results, we propose a way in which
the diffusible signal molecule Slit and the neuronally expressed receptor Robo
control the development of dendrites in the CNS of Drosophila
embryos.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Staging of embryos
Embryos were collected and incubated at 25°C. Developmental stages are
indicated in hours at this standard temperature.
Dimensions of embryonic CNS
The mean width of the CNS in the abdominal half-segments 3-5 of unfixed
wild-type embryos was 29, 41 and 43 µm at hours 9, 14 and 17, respectively.
The mean widths of the CNS in various other genotypes at hour 14 were: 41
µm in slit2/+, 35 µm in
slit2/slit2, 40 µm in
Sdc10608/Sdc10608, 39 µm in
robo4/robo4, and 48 µm in
comm5/comm5. Note that all dimensions are
calibrated for live whole-mount embryos. Dissection can stretch tissues, and
fixation can shrink them by up to 20%. Therefore, we compared dimensions in
the CNS both in actual measurements (µm) and in percentages of the main
axes, i.e. the medial-lateral axis (from the midline to the edge of the CNS at
the mid-point between the centers of the anterior and posterior commissures)
and the anteroposterior axis (from one segmental boarder to another). By using
the percentages, the measurements from all individual dissected and/or fixed
embryos were each calibrated against the live whole-mount embryos of the same
age and genotype.
Immunocytochemistry
Antibody raised against a fragment from the EGF repeats (aa1311-1480) of
Slit (mAb C555.6D, 1:300 dilution; Developmental Studies Hybridoma Bank)
(Rothberg et al., 1990) was
used to visualize extracellular Slit protein in embryos during hours 9 to 17.
This antibody cannot distinguish between the full-length form of the protein
and its potential proteolytic in vivo product
(Brose et al., 1999). However,
developmental western analysis indicated that over 92% of Slit remains
full-length during hours 10-14 (Fig.
1A,B). The cell membrane was left untreated (unpermeabilized) with
detergent throughout the entire immunological procedure. This excluded from
detection the pool of Slit protein that is either within the source cells or
internalized into neurons and other non-source cells. A parallel test with
antibody against the cytoplasmic molecule Pak (p21-activated kinase) (rabbit
IgG, 1:400 dilution; source: Y. Takagi, Fukuoka Dental College, Fukuoka,
Japan) confirmed that, without detergent treatment, immunocytochemistry at
this stage cannot effectively detect proteins within cells in the embryonic
CNS (Fig. 1C-F). To preserve
relative immunofluorescent intensities, control and experimental embryos were
fillet-dissected in parallel in insect saline and immediately fixed for 10
minutes in freshly prepared 4% paraformaldehyde in phosphate buffer (pH 7.4).
This allowed direct comparisons among the embryonic CNSs of different stages
and different genetic backgrounds. In all cases, slit/slit null
embryos were also processed for immunocytochemisrty in the same reaction
plate. Their average immunofluorescence intensity was used to define the
baseline for quantification of the Slit topography in other genotypes.
Preparations were rinsed in phosphate buffer for 30 minutes and blocked in
0.2% BSA (bovine serum albumin) in phosphate buffer (PBSB) with 5% normal goat
serum for 4 hours. Primary antibodies were added in PBSB with 1% goat serum
and incubated overnight at 4°C. Preparations were then washed in phosphate
buffer three times for 15 minutes each and blocked again for 15 minutes in
PBSB with 5% normal goat serum. Subsequently, fluorescently (FITC, TRITC or
Cy5) labeled secondary antibodies (Jackson Laboratories) were added for 2
hours, after dilution in PBSB with 1% goat serum, rinsed in phosphate buffer
for 45 minutes, then in 0.1% Tween 20 in PBS for 15 minutes, and mounted in
Prolong medium (Molecular Probes). Addition of Tween 20 at this concentration
reduced the background while cellular membranes remained largely intact.
FITC-conjugated HRP (horseradish peroxidase) antibody (Jackson Laboratories)
was used to determine the position of the neuropil and longitudinal axon
fascicles.
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Single-cell labeling
Individual aCC motoneurons were retrogradely labeled with the lipophilic
fluorescent dye DiI as described previously
(Furrer et al., 2003). Tracings
of these motoneurons were done on projected DiI fluorescent images
(Fig. 2C,D for an example).
Size of aCC dendrites
As a measurement of the dendrite size in aCC, the tips of dendritic
processes that extended more than 1 µm from the axon shaft were counted,
and their positions relative to the midline noted.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
addition, an available GAL4 driver (eve'-GAL4)
(Fujioka et al., 2003) was used
to achieve cell-specific expression of various transgenes in aCC. As with
other neurons in vivo (Kim and Chiba,
2004), the aCC undergoes a period of axogenesis before initiating
dendrogenesis. At hour 9 of wild-type embryogenesis, the aCC produces a single
axon that extends laterally toward its target, muscle-1, about 100 µm from
the aCC cell body. Beginning at hour 13, about 1 hour before the axonal growth
cone of the aCC contacts muscle-1, the aCC begins to develop its main
dendrites within the newly emerging neuropil, as collateral processes from the
CNS portion of its axon. At the same time, the aCC also extends a medially
directed growth cone that subsequently crosses the midline. This second growth
cone eventually develops into contralateral dendrites. In this study, we
focused on the first dendrites that the aCC develops ipsilaterally. By hour
14, the aCC has extended several filopodia-like dendritic tips along its axon
shaft (Fig. 3B,D). By hour 17,
the aCC has 15.1±1.5 dendritic tips centered 13.8 µm from the
midline (Fig. 3C,E). Such a
stereotypy of aCC dendrogenesis allows detection of abnormal development in
the CNS of various mutants.
Neuropils, the regions devoid of cell bodies but enriched with synapses,
are important landmarks in the CNS. In Drosophila embryos, as in many
other animals, the neuropils occurs as a bilateral pair in each segment where
midline-crossing commissures and longitudinal connectives intersect
(Fig. 3A). Starting at hour 9,
a neural surface marker, anti-HRP (Jan and
Jan, 1982), reveals gradual thickening of this area with neuronal
processes. After hour 14, the synaptic vesicle protein Synaptotagmin 1 begins
to be detected in the CNS and, by hour 17, it accumulates as puncta through
the whole width of longitudinal connectives but not in midline-crossing
commissures (data not shown). Nearly concurrently, dendrites of CNS neurons
develop within the longitudinal connectives. In our study, we refer to this
region from hour 14 onward as the neuropil, and use anti-HRP antibody to
estimate its width and position along the medial-lateral axis of the CNS.
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; Wolf and Chiba,
2000). [Note: Wolf and Chiba
(Wolf and Chiba, 2000) report
one rare incidence in robo/robo mutant embryos (out of 38 cases
examined) of the aCC abnormally extending its axon across the midline but then
following the normal but mirror-image contralateral pathway.] However, the aCC
in robo/robo embryos exhibits its first visible defects as it begins
to develop dendrites. At hour 17, the size of aCC dendrites, measured as the
number of dendritic tips, is reduced to 26% of the wild-type size
(Fig. 4A). Because aCC
development prior to the onset of dendrogenesis is normal in
robo/robo mutants, it offers a relatively straightforward means for
evaluating the role of Robo in dendrogenesis.
To determine whether Robo is required in the aCC during its dendrogenesis,
we designed two complementary tests that are bath based on single-cell genetic
manipulations. First, we expressed a robo RNAi construct
(Tayler et al., 2004)
transgenically in the aCC in a wild-type background
(eve'-GAL4RN2/UAS-roboRNAi). This cell-specific
RNAi against Robo in the aCC reduces its dendritic size to 34% that of wild
type. This reduction is similar (P=0.28 by two-tailed
t-test) to that observed in the robo/robo embryos
(Fig. 4C). Second, we
transgenically resupplied wild-type Robo protein to the aCC in
robo/robo embryos
(robo1/robo4;eve'-GAL4RN2/UAS-roboWT).
Even when surrounded by cells that lack Robo, this cell-specific rescue
reverts the dendrogenesis defects to almost wild type
(Fig. 4B,C). The sub-100%
rescue of the phenotype is not simply due to a low level expression of Robo
transgene product in the aCC (see below for a probable explanation). These
results indicate that Robo is both necessary and sufficient to
cell-autonomously support the dendrogenesis in aCC motoneurons.
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). This finding suggests a possible role of Slit during aCC
dendrogenesis. However, the time-course for neuropilar accumulation of Slit, a
critical parameter for establishing a causal relationship, is unknown.
Therefore, we examined the spatiotemporal concentration topography of
endogenous Slit, which the emerging axon and dendrites of aCC probably
encounter within the embryonic CNS. We used detergent-free immunocytochemistry
(see Materials and methods) in order to exclude (a) the pool of Slit protein
within the midline glia that is yet to be secreted, and (b) the pool that has
been internalized into neurons and non-midline glia and thus is no longer
capable of activating Slit receptors on the surface of the aCC and other
neurons in the CNS. This leaves only the pool of Slit protein that exists in
the extracellular space and, thus, represents the pool capable of signaling
various Slit receptors on the neuronal cell surface.
The results reveal a complex concentration topography of Slit within the
extracellular space of the CNS that dynamically changes through development
(Fig. 5A-D). We quantified Slit
in a defined three-dimensional space that roughly matches where the aCC axon
extends and its dendrites will develop
(Fig. 5, boxed region; also see
Fig. 2A,B). We chose three time
points: hour 9 before neuropil development, hour 14 immediately after the
onset of aCC dendrogenesis, and hour 17, nearly 4 hours after the process has
begun. Prior to the emergence of the neuropil, there is a gradient of Slit
that descends steeply from its midline source
(Fig. 5E). This is when the
growth cones of many axons orient themselves, each toward a specific direction
with respect to the midline (Furrer et al.,
2003; Kidd et al.,
1999; Wolf and Chiba,
2000). At this time, the distribution of Slit is similar to many
other diffusible signal molecules, as a descending gradient from its source.
However, once the neuropil begins to develop, a notable deviation occurs at
approximately 10 µm from the midline, where the longitudinal fascicles
begin to thicken. Slit starts to accumulate at this position. By hour 14, this
local accumulation reaches 43% of the midline level
(Fig. 5F). Subsequently, Slit
levels at both the midline and neuropils rise continuously. Neuropils begin to
emerge at the site of this secondary Slit accumulation. The aCC dendrites also
develop at the very site of the Slit enrichment and immediately after, but not
before nor long after, local Slit concentration begins to rise there. This
spatiotemporal coincidence raises the possibility that Slit at the neuropil is
either an inducer or facilitator of dendrogenesis in the aCC.
In the robo/robo mutant embryos, the overall amount of Slit protein at hour 14 is reduced to 79% of wild-type amounts (Fig. 6A,B), presumably reflecting the normal Robo function of capturing the protein on the cell surface and its contribution to the establishment of Slit topography. In addition, the secondary accumulation of Slit is medially shifted in the robo/robo embryos. This medial shift occurs in an exact parallel to that of the neuropil. aCC dendrites in the robo/robo embryos are also medially shifted, and are small when compared with wild-type dendrites (Fig. 4B). The shift in dendritic position matches those of both the neuropil and the local accumulation of Slit (Fig. 6C). As shown above, resupply of wild-type Robo to the aCC results in an increase in the dendritic size, to 70% of wild type (Fig. 4B,C). This reduction in dendritic size in the mutant parallels that of the volume of the local Slit peak (compare Fig. 6B with Fig. 6C). These results suggest that although Robo serves as a receptor detecting the presence of Slit, the amount of Slit at the neuropil modulates Robo activities.
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). Furthermore, slit/slit embryos survive until late
embryonic stages, thus providing an opportunity to test the idea that Slit may
play a role in dendrogenesis in the CNS. In Slit loss-of-function
(slit2/slit2) mutants, the aCC extends its axon
into the periphery (data not shown). Also, it produces a dendritic growth cone
that crosses the midline (Fig.
7B, arrowhead). However, the aCC fails to develop any collateral
dendrites along its axon (Fig.
7B,C). Motoneurons RP2 and U, too, generate a reduced number of
dendrites (data not shown). These results show that loss of Slit leads to a
loss of dendrites in the developing CNS.
Potential role of Slit at neuropil
Although dramatic, the loss of aCC dendrites in slit/slit embryos
(Fig. 7) might not indicate a
direct dendrogenic role for Slit. Even before the normal onset of aCC
dendrogenesis the CNS midline collapses in these mutant embryos
(Kidd et al., 1999;
Rothberg et al., 1990;
Sonnenfeld and Jacobs, 1994).
At the time dendrogenesis would normally occur, the CNS lacks a neuropil
(Fig. 5D). It is possible that
the emerging neuropil provides factors other than Slit that are responsible
for inducing dendrogenesis in the aCC. Alternatively, Slit and the
neuropil-derived factor(s) might both be required. To evaluate these
possibilities, we examined dendrogenesis in additional genetic
backgrounds.
The Commissureless loss-of-function mutation (comm5/comm5) shifts the neuropil laterally (Fig. 8A left). We reasoned that if the neuropil moves laterally, then the position of the dendrites would probably move laterally, as well. We found that aCC dendrites indeed form at the neuropil, their size being 56% that of wild-type neuropils (Fig. 8B,D). However, to our surprise, Slit is also found at 64% of the wild-type level in the laterally shifted neuropil (Fig. 8A right, also Fig. 8C). Thus, there is a three-way correlation among the neuropil, Slit and aCC dendrites in these embryos.
We next examined Slit heterozygous (slit2/+) embryos. With half the dose of the slit gene, embryos express Slit protein at 49% of the wild-type level, but otherwise keep the overall Slit topography with accumulation at the midline and the neuropil (Fig. 9A right, also Fig. 9E). Despite that, the animals are viable as heterozygotes, and their CNS and neuropil appears virtually wild type (Fig. 9A left). We reasoned that if the neuropil without a normal amount of Slit were sufficient, then the aCC would still develop normal dendrites at the neuropil. However, we found that the number of aCC dendritic tips in the slit/+ embryos dropped to 52% of the wild-type value (Fig. 9C,F).
Syndecan is another cell surface receptor known to bind Slit
(Johnson et al., 2004;
Steigemann et al., 2004). In
Syndecan (Sdc10608/Sdc10608) mutant
embryos, we noted a 51% loss of Slit from the neuropil as well
(Fig. 9B right, also
Fig. 9E). Nevertheless, the
neuropil, is relatively normal in size and position
(Fig. 9B left). aCC dendrites
in the Sdc/Sdc embryos develop within the neuropil
(Fig. 9D). However, as in the
slit/+ embryos, the number of aCC dendritic tips drops to 68% of
wild-type numbers (Fig. 9D,F).
Again, this drop corresponds fairly well to the reduction in the Slit
concentration at the neuropil. Thus, in both slit/+ and
Sdc/Sdc embryos, the neuropil remains more or less wild type, while
the size of aCC dendrites decreases in parallel to the amount of Slit present
at the neuropil.
Sdc/Sdc mutants also provide additional insights. Sdc/Sdc and robo/robo mutations reduce the size of aCC dendrites similarly, i.e. by 68% and 74%, respectively. However, whereas robo/robo embryos lose only 26% of neuropilar Slit, Sdc/Sdc embryos lose 60%. These observations are consistent with the idea that Syndecan serves as a co-receptor, that presents Slit to other Slit receptors expressed at the neuropil, whereas Robo functions as a receptor mediating the dendrogenic role of Slit.
These results lead to two conclusions. First, although contributions of yet-to-be-identified neuropil-derived factors cannot be ruled out, both the timing and the relative extent of dendrogenesis are positively correlated to the dynamic presence of Slit at the neuropil. Second, while different receptors, including Robo and Syndecan, can trap Slit protein on the cell surface, it is Robo that is likely to play a major role in signaling with localized Slit during dendrogenesis.
Slit alone is sufficient to induce dendrogenesis
Would presentation of Slit alone be sufficient to induce dendrogenesis? To
answer the question, we expressed full-length wild-type Slit in muscle-12,
outside the CNS, using a muscle-12-specific GAL4 driver
(GAL4M12/UAS-slitWT) (see
Fig. 3A). The aCC and other ISN
(intersegmental nerve) motoneuron axons
normally extend beyond muscle-12, before this GAL4 driver starts transgene
expression. Normally, these axons are in contact with muscle-12, but do not
generate any collateral processes here. Overexpression of Slit in muscle-12
fails to induce any collateral processes (0%, n=29 neurons) from any
of these axons (Fig. 10A,B
circle). By contrast, the ectopic Slit stalls the growth cone of the RP5
motoneuron from its target muscle-12 (100%, n=27 neurons)
(Fig. 10B arrow). These
results give further support to the idea that presentation of Slit alone is
not sufficient to induce collateral dendrogenesis from the aCC along its
axon.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wang et al., 1999;
Ward et al., 2005;
Whitford et al., 2002). Our
goal in this study was to examine the role of Slit and Robo in the context of
in vivo dendrogenesis in the CNS. Dendrogenesis is a late-stage event in the
differentiation of neurons. Thus, to uncover the specific role of molecules
responsible for dendrogenesis, one must not only demonstrate their
loss-of-function phenotype but also isolate their cell-autonomous operation.
Furthermore, it is also necessary to uncouple the earlier contribution of the
molecules, to either neurogenesis or axogenesis, from their direct
contribution during dendrogenesis. We chose to focus on the aCC motoneuron,
one of the first CNS neurons to generate dendrites in Drosophila
embryos and also one that can be genetically manipulated and visualized at the
single-cell level. Our results support the conclusion that in neurons Slit,
signaling through Robo, is responsible for controlling the timing,
positioning, and size of dendrites in the embryonic CNS. They also offer
insights into the complexity that surrounds the development of dendrites in
vivo.
Single-cell analysis on the role of Robo
In robo/robo embryos, the aCC produces small dendrites
(Fig. 4B). It is possible that
this residual dendrogenesis reflects partial functional redundancy among Robo
family receptors. RNAi against the robo gene in the aCC also results
in small dendrites (Fig. 4C).
Conversely, cell-specific resupply of wild-type Robo in the aCC reinstates its
ability to grow dendrites (Fig.
4C). These results, together with the fact that the aCC neurons in
robo/robo embryos have no other defects prior to the onset of
dendrogenesis, support the specific role of Robo in dendritic development.
The dendrogenic role of Robo was first demonstrated by Ghosh's group
(Whitford et al., 2002). Their
key experiment was inhibition of neurite branching in cultured neurons through
overexpression of the cytoplasmically truncated Robo. In our hands, similar
attempts to use a Drosophila version of cytoplasmically truncated
Robo have failed to induce any extra or abnormal dendrogenesis in vivo (data
not shown). Instead, we show that both genetic deletion and RNAi against the
robo gene cause dendrogenesis defects in uniquely identified CNS
neurons (Fig. 4). The
difference in effectiveness of dominant-negative proteins between the
mammalian and Drosophila neurons might simply reflect whether or not
Robo is a rate-limiting factor in a given neuron. Alternatively, data from
both Ghosh's group and ours are consistent with Robo being required
cell-autonomously during dendrogenesis.
Robo is expressed throughout neuronal development, not just during the
period of axon guidance analyzed by the majority of in vivo studies to date.
Previously, single-cell analyses in the embryonic Drosophila CNS have
shown a role for Robo in directing growth cones away from the Slit-secreting
midline. Without Robo, the axons of RP2 motoneurons are misguided medially
(Wolf and Chiba, 2000). Later,
the same Robo-lacking RP2 neurons also misguide their dendritic growth cones
towards the midline (Furrer et al.,
2003). In comparison to RP2, aCC motoneurons do not normally rely
on Robo to properly orient axonal and dendritic growth cones. However, when
Robo is overexpressed in the aCC, its dendritic growth cone can be made to
avoid the midline (data not shown). In all these cases, it would appear that
Robo causes growth cone collapse upon detecting Slit at the midline. By
contrast, our study supports a role for Robo in promoting the formation of
collateral dendritic processes. aCC motoneurons cell-autonomously require Robo
during dendrogenesis (Fig. 4).
Clearly, the same receptor has distinct roles, either collapsing growth cones
or promoting collateral dendrogenesis, i.e. two seemingly opposite types of
cellular responses, sometimes even within a single neuron. Although we do not
yet know the underlying mechanism, it is intriguing that migrating myoblasts
also exhibit a developmentally regulated response switch of Slit-Robo
signaling from repulsion to attraction in Drosophila embryos
(Kramer et al., 2001).
Insights from Slit topography
The Slit concentration topography of the embryonic CNS exhibits a dynamic
four-dimensionality (Fig.
5E,F). Previously, it was postulated that there is a descending
gradient of Slit from its source (Goodhill,
2003; Rajagopalan et al.,
2000; Simpson et al.,
2000). Indeed, both in culture media and within imaginal discs,
diffusible signaling molecules set up gradients that descend from their source
(Tabata, 2001;
Tessier-Lavigne et al., 1988).
We find that the actual Slit topography in the embryonic CNS is much more
complex (Fig. 5). Unlike
culture media, the embryonic CNS redistributes molecules such as Netrin
(Brankatschk and Dickson, 2006;
Hiramoto et al., 2000) and
Slit (this study) from their original source. Already by hour 14, the time
when the first dendrites begin to form, a prominent secondary accumulation of
Slit is present locally 10-20 µm away from the midline source of Slit
(Fig. 5F arrow). The local
concentration is approximately 43% of that at the midline, and the amount of
Slit that is found beyond 10 µm from the midline, the local minimum, is 56%
of the total extracellular Slit in the whole CNS.
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) or direct filopodia-mediated delivery
(Vasenkova et al., 2006). Once
there, Syndecan plays a role in capturing the extracellular Slit
(Johnson et al., 2004) (this
study). Our study suggests that the presence of Robo at the neuropil also
contributes to Slit capture on cell surface
(Fig. 6B). In addition to
Syndecan and Robo, at least two more Slit receptors, Robo2 and Robo3, are
known in Drosophila. When bound to such receptors on the surface of
migrating axons, Slit could be transported along commissural and longitudinal
fascicles. Individual Robo receptors are expressed in overlapping but distinct
sets of neurons (Rajagopalan et al.,
2000; Simpson et al.,
2000). Plenty of molecular heterogeneity and cellular dynamics
exists within the developing nervous system that could contribute to an
extensive redistribution of Slit within the CNS.
We propose that neural development utilizes the complex Slit topography to
control dendrogenesis. First, the position of the aCC collateral dendrogenesis
coincides with the local Still accumulation. Except for the slit/slit
embryos where there is no Slit present, all other genetic backgrounds examined
in our study have aCC dendrites developing where Slit accumulates locally.
Second, there is a positive correlation between the size of aCC dendrites and
the amount of Slit present. A notable exception is the robo/robo
embryos, in which the size of aCC dendrites is attenuated due to the loss of
Robo, a Slit receptor, in the neuron (Fig.
4). In Drosophila embryos, evidence for Slit proteolysis
has been presented by Brose et al. (Brose
et al., 1999). Because the Slit antibody recognizes the carboxyl
terminus region of the protein, it does not distinguish between full-length
Slit, which is capable of activating Robo, and the carboxyl-terminus fragment
of the proteolytic product, which is not. We have assessed the developmental
control over Slit proteolysis. Our quantification shows that, at hour 14, the
proteolysis affects only about 8% of the total volume of Slit
(Fig. 1B). Independent data
also suggest that Slit at the neuropil and beyond is indeed in the form that
is capable of stimulating Robo (I.V., M.-P.F. and A.C., unpublished). In our
study, we assume that a majority of Slit protein detected by the antibody in
the neuropil is in the full-length form, and take the positive correlation
between the Slit profile and aCC dendrogenesis to suggest that Slit acts in an
instructive role, setting the size of dendrites. Third, the time at which Slit
begins to accumulate at the emerging neuropil immediately precedes the
initiation of collateral dendrogenesis in the aCC. This indicates that Slit
accumulation is not simply a consequence of dendritic development. Instead,
the tight spatiotemporal correlation between Slit topography and aCC
dendrogenesis supports a model in which Slit plays a crucial role.
The phenotype of slit/slit embryos
The slit/slit phenotype during the period of dendrogenesis is
dramatic. Visualization with the anti-HRP antibody and retrograde DiI labeling
in late-stage slit/slit embryos reveals that many motoneurons extend
out axons in the CNS without Slit. Yet, they fail to initiate dendrites. Thus,
the phenotype that motoneurons such as aCCs
(Fig. 7B) and RP2s (data not
shown) exhibit is unique. However, there is a problem in attributing a direct
cause of the dendrite-less motoneurons to the absence of Slit. This is because
slit/slit embryos form very few axon fascicles, resulting in a
virtually neuropil-less CNS (Fig.
5D). Therefore, the dendrogenesis defects observed in
slit/slit embryos could be accounted for by any of the following
three scenarios: (1) the neuropil, not Slit, induces dendrogenesis, (2) Slit
alone is required, or (3) both the neuropil and Slit are required. Of these,
the third scenario is the most likely (see below).
In vivo complexity that impacts dendrogenesis
Hints about the additional factors that impact dendrogenesis are available
not only where neurons develop dendrites, but also where they do not. Except
for slit/slit, all other genotypes examined in our study develop a
neuropil in the CNS. In all cases, the aCC forms collateral dendrites at the
neuropil, but not anywhere else. However, no neuron that extends its axon or
dendrite across the midline develops dendritic branches at the midline despite
the fact that the midline is the sole source of Slit in the CNS
(Furrer et al., 2003;
Landgraf et al., 1997;
Landgraf et al., 2003).
Furthermore, we find that, unlike dissociated neurons in culture
(Whitford et al., 2002),
ectopic Slit presented outside of the CNS, at muscle-12, does not induce
collateral dendrogenesis in aCC motoneurons
(Fig. 10). What are the
factors that spatially restrict the dendrogenic function of Slit-Robo
signaling to the neuropil? It is possible that such factors are present at the
neuropil itself, serving a permissive role. However, it is also conceivable
that the active suppression of dendrogenesis occurs outside the neuropil,
including at the CNS midline and outside the CNS. In addition to such
extrinsic factors, each neuron could display intrinsic molecular biases
towards a certain portion of its axon. If this were true, then one might
anticipate finding mutations that cause reduced dendrites at the neuropil, as
well as mutations that cause ectopic collateral dendrogenesis outside the
neuropil. Recently, we have found several mutants that fit both of these
categories (our unpublished results). Characterization of these mutations will
not only help identify additional factors that impact dendrogenesis, but also
offer insights into the general question of how spatiotemporal precision in
dendrogenesis is regulated within the CNS.
|
|
;
Erskine et al., 2000;
Hao et al., 2001;
Kidd et al., 1999) and
coordinates positioning of longitudinal fascicles
(Rajagopalan et al., 2000;
Simpson et al., 2000). As long
as the midline peak persists, it continues to repel dendritic growth cones
(Furrer et al., 2003). However,
at the emerging neuropil, the concentration of extracellular Slit also rises
steadily, creating a second Slit-enriched region within the developing CNS.
Here, Slit-Robo signaling has an additional role as a promoter for
dendrogenesis. Thus, the same Slit-Robo signaling that repels growth cones
from the midline, also produces dendrites at the neuropil, thereby sculpting
the neural architecture at multiple stages. How various molecules that are
known to impact dendritic morphology (Gao
and Bogert, 2003; Grueber and
Jan, 2004) may be linked to Slit-Robo signaling remains an open
question. Future study is needed to address how Slit and its receptor Robo
collaborate with diverse signaling partners at multiple steps of neural
development, to serve as the `architects' of the developing CNS. |
ACKNOWLEDGMENTS |
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|
Footnotes |
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