Axon fasciculation and differences in midline kinetics between pioneer and follower axons within commissural fascicles
Magdalena Bak and
Scott E. Fraser*
Division of Biology, Biological Imaging Center, Beckman Institute,
California Institute of Technology, Pasadena, CA 91125, USA
*
Author for correspondence (e-mail:
sefraser{at}caltech.edu)
Accepted 1 July 2003
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SUMMARY
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Early neuronal scaffold development studies suggest that initial neurons
and their axons serve as guides for later neurons and their processes.
Although this arrangement might aid axon navigation, the specific
consequence(s) of such interactions are unknown in vivo. We follow forebrain
commissure formation in living zebrafish embryos using timelapse fluorescence
microscopy to examine quantitatively commissural axon kinetics at the midline:
a place where axon interactions might be important. Although it is commonly
accepted that commissural axons slow down at the midline, our data show this
is only true for leader axons. Follower axons do not show this behavior.
However, when the leading axon is ablated, follower axons change their midline
kinetics and behave as leaders. Similarly, contralateral leader axons change
their midline kinetics when they grow along the opposite leading axon across
the midline. These data suggest a simple model where the level of growth cone
exposure to midline cues and presence of other axons as a substrate shape the
midline kinetics of commissural axons.
Key words: Forebrain, Zebrafish, Commissural axon, Neuronal scaffold, Growth cone, Intermediate target, Growth cone morphology, Growth cone behavior, GATA2, Axon pathway
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Introduction
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During development of the central nervous system (CNS), neurons
differentiate and connect with one other, enabling information processing and
the establishment of specific behavioral patterns. A better understanding of
the early events in neuronal connectivity in the forebrain is a fundamental
step in understanding how the brain assembles itself and functions. Unlike
their adult counterparts, early embryonic brains are relatively simple, with a
small number of neurons and neuronal tracks, offering unique advantages for
mechanistic studies. Detailed studies of tract formation in invertebrate
nervous systems have shown that a small number of early neurons, termed
pioneers, lay down an axonal scaffold that later axons and their growth cones
follow (Bate, 1976
;
Bentley and Keshishian, 1982
;
Bastiani et al., 1984
;
Jacobs and Goodman, 1989
;
Boyan et al., 1995
). Removal of
these pioneers adversely affects the pathfinding of later axons
(Raper et al., 1984
;
Klose and Bentley, 1989
;
Gan and Macagno, 1995
;
Hildalgo and Brand, 1997). Growth cones of pioneer and later axons differ in
their morphology: early axons posses larger and more complex growth cones
(LoPresti et al., 1973
;
Bastiani et al., 1984
). And
growth cone morphology changes correlate with specific choice points along the
axon route (Myers and Bastiani,
1993
).
Analogous mechanisms may be involved in establishing the initial neuronal
circuitry in vertebrates. For example, connections between the cerebral cortex
and the thalamus originate from a transient population of subplate neurons
that establish this connection during embryonic stages
(McConnell et al., 1989
).
Using fluorescent lipophilic dye tracers, growth cone morphology differences
between these early axons and later axons found in this projection, have been
observed in ferrets and cats (Kim et al.,
1991
). Growth cone morphology has been correlated with axon
decision making, especially at choice points such as the optic chiasm and the
floor plate. In these regions, growth cone morphology and sometimes growth
behavior significantly change, with pioneering growth cones assuming complex
shapes while later axons have more streamline growth cones
(Bovolenta and Dodd, 1990
;
Wilson and Easter, 1991; Mason and Wang,
1997
). Similar to invertebrate systems, the earliest axons in
vertebrates are in the right place and at the right time to establish initial
projections that later axons can then follow. For example, in zebrafish,
secondary motoneurons in the absence of primary motoneurons are not able to
correctly pattern dorsal nerves but do form correct, albeit delayed, ventral
projections (Pike et al.,
1992
). Taken together, these data support the hypothesis that
initial axons might play a pioneer-like role in establishing later axonal
tracts, perhaps similar to their role in invertebrates. In vivo time-lapse
analysis of axonal tract formation is needed to directly address how the
initial axons affect later axons during early neuronal scaffold formation.
At one day of development, the zebrafish forebrain neuronal scaffold
contains several distinct bilaterally symmetrical clusters of neurons,
interconnected by a limited number of neuronal tracts and commissures
(Chitnis and Kuwada, 1990
;
Wilson et al., 1990
;
Ross et al., 1992
)
(Fig. 1B), analogous to the
early neuronal scaffold of mouse embryos
(Mastick and Easter, 1996
).
During the next few days, the scaffold increases in size as more neurons and
axons are added to the existing tracts, but few new axonal tracts appear. For
example, initially the postoptic commissure (POC) connects two clusters of
ventral neurons (the left and right ventrorostral cluster; vrc) in the
forebrain (Ross et al., 1992
)
(Fig. 1B) with additional axons
from other brain regions projecting across it at later stages (Wilson and
Easter, 1991). In zebrafish, cell labeling methods in combination with
surgical manipulations reveal that early brain tracts aid in axon guidance for
initial axons of other tracts (Chitnis and
Kuwada, 1991
; Wilson and Easter, 1991;
Chitnis et al., 1992
). Whether
early axons influence later axons inside the same axon fascicles has not been
addressed.

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Fig. 1. Zebrafish forebrain primary neuronal scaffold contains gata2::GFP-positive
neurons. (A) 24 hpf zebrafish transmitted light view. The approximate location
of the early neuronal clusters in the forebrain are schematically shown. (B)
Schematic drawing illustrating the position of the major neuronal clusters and
the axon tracts that connect them in the anterior CNS in zebrafish (based on
Chitnis and Kuwada, 1990 ;
Wilson et al., 1990 ; Ross et
al., 1991). (C,D) Confocal images of dorsal (C) and lateral (D) views of a 24
hpf gata2::GFP zebrafish showing the location of GFP-positive cells in
relation to forebrain morphology. The cells are located bilaterally (C) and
occupy the rostroventral part of the diencephalon along the optic recess (or)
(D). (E,F) Fluorescent confocal images showing the vrc stained with acetylated
alpha tubulin antibody (red) to reveal the identity of gata2::GFP-expressing
cells (green). White asterisk indicates a vrc cell with little or no GFP;
black asterisk marks a GFP-positive vrc cell. White arrows indicate cells
where colocalization of the neuronal antibody and the GFP can be seen (E). At
24 hpf all vrc cells express GFP and appear yellow due to spectral overlap
between the green GFP and the neuronal antibody (F). Scale bar: 80 µm in A;
20 µm in C; 10 µm in D-F. tt, telencephalon; dc, diencephalon; drc,
dorsorostral cluster; vrc, ventrorostral cluster; vcc, ventrocaudal cluster;
ec, epiphysial cluster; SOT, supraoptic track; TPOC, track of postoptic
commissure; POC, postoptic commissure; AC, anterior commissure; or, optic
recess.
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Here we build upon studies of the retinotectal projection
(Hutson and Chien, 2002
),
olfactory neuron pathfinding (Dynes and
Ngai, 1998
), and growth cone and dendrite dynamics of spinal cord
motoneurons (Jontes et al.,
1999
) to examine the role of early axon guides on midline kinetics
of commissural axons. Using a stable transgenic zebrafish line
(Meng et al., 1997
) and
timelapse confocal laser-scanning microscopy we reveal a new level of
complexity in commissural axon kinetics at the midline. Our data shows that
midline kinetics in vertebrate commissural axons result from the combination
of highly adaptive axons, dynamics interaction between them and the different
exposure of each growth cone to other axons and the local cues.
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Materials and methods
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Fish maintenance
Raising and spawning of adult zebrafish were performed as outlined in the
Zebrafish book (Westerfield,
1995
) and in accordance with the guidelines of the California
Institute of Technology.
Whole-mount immunohistochemistry and confocal microscopy
Embryos at stages 19-27 hpf were fixed, blocked and incubated with primary
antibodies as previously described (Wilson
et al., 1990
). Acetylated alpha tubulin antibody (Sigma) was used
to label neuronal cell bodies and their axons
(Wilson et al., 1990
;
Ross et al., 1992
). For some
experiments, a cocktail of neuron specific antibodies zn1,8,12 (Developmental
Studies Hybridoma Bank) was used for neuronal cell body labeling. The neuron
specific antibodies were detected with secondary TRITC antibody (Jackson Lab).
Inherent GFP was imaged. Embryos were deyolked prior to imaging and the head
region (anterior to hindbrain) was dissected for imaging. Embryo heads were
placed in bridge slides, covered with coverslips and imaged using the Zeiss
LSM510 laser-scanning confocal microscope at 40x.
Timelapse confocal microscopy
Embryos at 20-22 hpf were anesthetized with tricane in sedative amounts
(0.01%) and embedded in a drop of 1-1.2% ultralow melt agarose on a cover
slip-bottom petri dish in 30% danieau/0.01%tricane/0.15 mM PTU (to bleach
pigment). Imaging was performed using inverted Zeiss Pascal confocal with a
Plan-Neuofluar 40X/N.A1.3 oil objective as well as Zeiss 510 confocal with
Achroplan IR 40x/0.8 W, 63x/0.9W and C-Apochromat 40x/N.A
1.2 objectives. Three-dimensional stacks of the forebrain were taken at 6, 3
and 1.5 minute intervals spanning the vrc and the POC. Temperature was
maintained at 28-29°C throughout all imaging. GFP-positive cells were
excited with the 488 nm argon laser line using 505LP chroma filter set.
Typical imaging experiment (n=22 separate live speciments) lasted
between 3-5 hours. A z-stack spanning approximately 60 µm was
collected at each time point with individual sections being 1 µm apart. The
pinhole settings were at 2.0-2.77 airy units. Refocusing was minimal but
needed to be done occasionally to make sure the growth cones were imaged in
full. z-stack images were imported into Object Image and maximum
intensity projections (MIPs) were made at each time point. These were later
assembled into movies.
Axon growth rates analysis
Time-lapse data were analyzed with a 4D visualization software (Slidebook
Intelligent Imaging Innovations), which allowed us to import our movies from
Object Image and trace individual axon lengths at each time point during the
time-lapse. Midline was found using a transmitted light image from each
timelapse. Axons were traced, measured and plotted as variation in position
(distance) from midline along the arc of the POC trajectory
(Fig. 3G). Axon lengths were
also used to compute average growth rates. All axon length data were divided
into two groups: axons with no commissure [i.e. leading axons (n=16)]
and those where a commissure was already in place [i.e. follower axons
(n=24)]. For each axon, the average growth rate±s.e.m at
midline, defined as ±10µm on each side of the midline was
calculated. The growth rate was compared to the average growth
rate±s.e.m. outside the midline for both axon groups. To confirm that
our sampling time interval was adequate, timelapses were carried out at 1.5, 3
and 6 minute intervals and the numbers were analyzed separately for each
group. Because all data were consistent across the different sampling
frequencies, they were pulled together for final analysis.

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Fig. 3. DiI filled gata::GFP-positive vrc cluster where first GFP axon has just
started to grow to midline shows no red axons ahead of the GFP. gata::GFP
embryo fixed immediately after the first GFP-positive axon began growing to
midline and the vrc cluster (asterisk) was filled with DiI to label axons from
the vrc. (A) Front view of the green channel showing the GFP-positive vrc
cells and the first GFP-expressing growth cone (arrow) projecting towards the
midline. (B) Merged green and red emission channels showing the injected vrc
cluster (asterisks) and the labeled growth cone (arrow). No other DiI filled
axons are visible ahead of the GFP-expressing axon. (C) Red channel showing
the DiI fill and the labeled DiI growth cone (arrow). Scale bar: 10 µm.
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DiI labeling
Gata::GFP embryos were prepared for timelapse experiment and imaged until
the first GFP-expressing growth cone appeared. The embryos were removed and
immediately fixed in 4% PFA for 1.5 hours at room temperature. Fixed embryos
were remounted in 1% agarose in PBS and injected under fluorescent microscope
with DiI into the GFP-positive vrc cluster in order to label axons from the
vrc cells. DiI-injected embryos were incubated at room temperature for 12-24
hours to allow the dye to diffuse throughout the axons and growth cones.
Embryos heads were removed and remounted in agarose for analysis. Imaging was
carried out using Zeiss Pascal inverted confocal scanning microscope, using
488 nm and 543 nm excitation sequentially using multitrack mode and a
C-Apochromat 40x/1.2 water objective.
Growth cone morphology analysis
The highest quality time-lapses were selected for this analysis. Growth
cone morphology was analyzed using ImageJ measurement and analysis software.
Growth cone areas were measured by outlining the growth cone excluding
filopodia. For width/length ratio measurements, the lengths of the growth
cones were measured from tip of the leading edge of the lamelipodium to the
base of the growth cone tip along a trajectory of the proximal region of that
growth cone's axon. The width was measured in each case as the perpendicular
segment positioned at the widest point for each growth cone. The average
growth cone area±s.e.m. and w/l ratio value were calculated and
compared between leading and follower growth cones. The number and lengths of
filopodia were recorded for each time frame of a movie for leading axons
(n=10) and follower axons. In the later case only axons with clearly
discernible filopodia were chosen (n=8).
Data analysis
Quantitative axon growth rates data was analyzed to test for significant
differences using Student's t-test analyses with Origin software. In
cases where one average growth rate was compared with two different growth
rates the P value was corrected for multiple comparisons.
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Results
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gata2::GFP labels a specific neuronal cluster of the forebrain
Neuronal differentiation and axonogenesis in the zebrafish forebrain have
been shown to begin between 17-28 hpf
(Chitnis and Kuwada, 1990
;
Wilson et al., 1990
;
Ross et al., 1992
). The
relative arrangement of the early neuronal clusters inside the brain and their
tracts, deduced from antibody staining against the neuron specific acetylated
alpha tubulin (Chitnis and Kuwada,
1990
; Wilson et al.,
1990
; Ross et al.,
1992
), is schematically illustrated
(Fig. 1A,B). To permit
reproducible in vivo imaging, we employ a stable transgenic line in which the
cis-regulatory domain of the transcription factor GATA2 drives GFP
(gata2::GFP) (Meng et al.,
1997
), and shows early expression in the earliest differentiating
cells in the forebrain (Fig.
1C,D). The GFP-positive cells, 8-10 µm in diameter, form
bilateral clusters in the ventral forebrain that extend along the optic recess
from the anterior diencephalon towards the ventral flexure
(Fig. 1C,D). Staining with
acetylated alpha tubulin antibody at 18-28 hpf reveals two distinct clusters
of acetylated alpha tubulin-positive ventrorostral cluster (vrc) neurons
(red); many of these cells express GFP (green)
(Fig. 1E). By 24 hpf, all vrc
neurons express GFP (Fig.
1F).
The first axons to form the POC are gata2::GFP positive
Immunohistochemistry with the acetylated alpha tubulin (AT) antibody shows
that the postoptic commissure (POC) is set up between 21 and 23hpf
(Fig. 2A-D). In addition to
labeling the axons of the early neuronal scaffold, AT also labels the cell
bodies of cells undergoing axonogenesis, ependymal processes and cilia
(Chitnis and Kuwada, 1990
), as
well as the superficial network of nerve fibers running just beneath the
ectoderm. At the early stages of POC formation these labeled processes are
closely juxtaposed and can appear as axon-like processes. Thus, our detailed
analyses of the earliest POC axons were performed on individual optical
sections.

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Fig. 2. gata2::GFP-positive axons pioneer the POC and also mark the majority of
later POC axons. Gata2:GFP cells and their axons are depicted in green and the
primary neuronal scaffold in red. Rostral is towards the left and dorsal is
towards the top. Images are maximum intensity projection (MIP) views of
confocal z stacks. (A-D) Lateral images at 21, 23, 25 and 27 hpf
showing the relative position of gata2:GFP cells (green) with respect to the
neuronal scaffold revealed with acetylated alpha tubulin (red). (E-H)
Corresponding frontal views of the POC show gata2::GFP-expressing axons
pioneer the POC. At 21 hpf, only early neurons differentiating away from the
neuroepithelium can be seen, but no axons are present where the POC will form
(broken white line) (E). At 23 hpf, a small number of axons can be seen across
the POC; these early axons express GFP although because of fixation some of
the GFP signal is too weak to be visible in the overlay image (F). At 25 and
27 hpf, respectively, the POC thickens. GFP-positive axons can be seen
spanning the entire width of the commissure (G,H). The small number of axons
that appear to not express GFP reflects axons from other brain regions that
project their axons along this tract. Scale bar: 20 µm.
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Double-labeling studies show that these earliest axons of the POC, detected
by AT, are GFP positive. At 21 hpf, no axonal staining with either AT or GFP
can be seen along the future POC trajectory except for the cell bodies and
ependymal processes of delaminating neuroepethelial cells
(Fig. 2E). At 23 hpf a small
number of axons slightly beneath the surface ectoderm are visible across the
POC (Fig. 2F). Analysis and
rendering of these double labeled preparations is often challenging because
the GFP expression levels in axons are weak after fixation. To confirm that
all of the initial axons growing across the POC are GFP positive, we employed
spectral analysis using a Zeiss LSM-510 META microscope. Given the low levels
of GFP expression, some axons appear reddish in the double overlay, even
though they express distinct GFP fluorescence. Analysis of the individual
z sections further confirms that both ependymal processes and
superficial nerve fibers extend near the commissural axons and these do not
express GFP. At later stages, more GFP positive axons appear
(Fig. 2H), as well as a small
number of axons that do not express GFP, probably originating from other
neuronal clusters as previously described (Wilson and Easter, 1991).
DiI-labeling experiments were performed to confirm that the first GFP axon
is the first axon along the future POC tract. Transgenic gata::GFP fish were
timelapsed until the first GFP-expressing growth cone appeared
(Fig. 3A), at which time the
embryos were fixed briefly to retain GFP and growth cone morphology. DiI
injection into the vrc cluster (Fig.
3C) reliably labeled the first GFP-expressing axon including its
growth cone; no DiI-labeled axons were visible ahead of the leading
GFP-positive process (Fig. 3B).
Thus, the first GFP-positive axon is the initial axon that grows along the POC
trajectory.
Axon kinetics during POC formation in vivo
To characterize the growth behavior of POC axons, we performed a series of
time-lapse confocal microscopy experiments (n=22) using gata2::GFP
zebrafish embryos. At 22-23 hpf, one to three discernable axons from the
bilateral vrcs advance rostrally along the POC path
(Fig. 4A). Within 1 hour, these
axons meet, creating a continuous axon arc along the future POC trajectory
(the early axon from the right is indicated with pink arrow in
Fig. 4A,B). Later axons from
both sides (blue arrows in Fig.
4A-D) fasciculate with the initial axons at various points along
the commissure and follow them across the midline (see Movie 1 at
http://dev.biologists.org/supplemental/).

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Fig. 4. Timelapse imaging of a wild-type gata2::GFP growth cones crossing the
ventral forebrain and forming the POC. (A-D) Selected images of single time
points from one 3 minute interval timelapse sequence showing a typical leading
growth cone (pink arrows in A and B) from the vrc cluster navigating towards
and past the midline, where it is joined by the growth cone from the opposite
cluster (not visible in B because it is masked by fluorescence from more
dorsal sections). Subsequently, later growth cones (blue arrows in B-D) also
cross the midline and grow across the POC. The midline is indicated by a
broken line; time is shown in minutes. Scale bar: 10 µm. (E) A typical
distance from midline along the POC trajectory axon plot. Axon length at each
time point is plotted as distance from midline along the POC trajectory. The
leading growth cone is plotted in pink and a later (follower) axon is plotted
in blue corresponding to the growth cones marked with pink and arrows in A-D.
The axons shown here start from the right vrc (top of the plot) and cross over
to the left vrc (bottom of the plot) as indicated in the schematic to the left
of the graph. The two axons advance across the midline with different rates as
indicated by the slope of each line (see pink and blue boxed regions). On the
right, two microscope configurations show how the imaging was performed. The
timelapse shown in A-D was obtained in the inverted configuration.
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These movies reveal two distinct classes of behaviors. The first axon that
emerges maintains its leading position towards the midline and across it
(Fig. 4A,B, pink arrow); the
trailing axons stay close behind, crossing the midline after the first axon
crosses (Fig. 4C,D, blue
arrow). To analyze the dynamics of axons along the POC trajectory, length and
position along the POC trajectory was measured at every time point for each
axon visible in our timelapse recordings. To allow easier comparison of axon
behaviors, axon growth was plotted as distance from midline along the POC
trajectory as a function of time (Fig.
4E). At the midline, the first visible axon (pink line) has an
almost flat slope, signifying that this axon slows down in this region (pink
boxed area). By contrast, a later axon (blue line) has a continuous, steep
slope at the midline (blue boxed area) showing that it does not slow down.
Thus the two types of axons show different behaviors at the midline, an
important intermediate target for commissural axons
(Kaprielian et al., 2001
).
POC axon nomenclature
In view of the order in which the axons reached and crossed the midline, we
have defined the first axon from either side to cross the midline as a leader
axon; all later axons observed along the POC are defined as follower axons. In
25% of the cases, axons originating from the two opposite vrcs arrived at the
midline at approximately the same time and both were defined as leader axons.
In the rest of the cases, one axon arrived early at the midline and grew
across it. In these cases, the initial axon was defined as a leader and the
contralateral axon was defined as a follower.
Quantitative analysis of leader and follower axons reveals
differences in their behavior at the midline
Quantitative analysis of POC axons (n=46) reveals a behavioral
difference between leader and follower axons in the midline region (±10
µm from the midline). Within this region, leading axons slow significantly
(Fig. 5A) compared with
follower axons (Fig. 5B),
correspondingly their average slopes in this region are markedly different.
This difference is robust: the difference between leader and follower axon
behavior at the midline is maintained even when they are averaged
(Fig. 5C). The extended time
that leading axons spend within the midline region is due to a more than 50%
reduction in growth by the leader axons at the midline. At the midline, leader
axons grow at an average rate of 35±5 µm/hour as opposed to
85±5 µm/hour, the average growth rate of leader axons along the rest
of the POC trajectory (Fig.
5D). Follower axons do not display this behavior; their average
growth rates inside (91.6±7.1 µm/hour) and outside (93.7±6.2
µm/hour) are equivalent (Fig.
5D). Thus, leader and follower axons grow at comparable rates
outside the midline region, but display drastically different growth rates
near the midline.

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Fig. 5. POC axon dynamics with respect to the midline. Quantitative analysis of
axon growth rates reveals a difference in axon behavior around the midline not
readily apparent from direct observation of timelapse experiments. (A,B)
Representative plots of distance from the midline along the POC trajectory
versus time for leader (A) and follower (B) axons. Leader axons spend longer
time within ±10µm of midline (broken black line in A-C), while the
growth of later axons does not slow down in this region. (C) Average behavior
of leader (n=16) and follower (n=24) axons at the midline.
Leader and follower axon plots were centered on the timepoint when each axon
crossed the midline. The average plot shows a more than twofold difference
between the time leader and follower axons stay at the midline. Individual
plots of all axons from the left vrc were reflected around the
x-axis. (D) Average growth rates±s.e.m. for leader (pink) and
follower (blue) axons with respect to the midline and outside midline region.
Leading axons grow significantly more slowly at the midline compared with
their average growth rate away from midline and compared with follower axons
at the midline (P<0.001, Student's t-test corrected for
multiple comparison).
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Both slower speeds and higher frequency of retractions contribute to
lower average growth
Axon growth rate reflects both the absolute speed and direction of growth.
Thus, two factors could contribute to the apparent slowing of leader axons at
the midline: (1) a decrease in axon growth speed and (2) higher number of
growth cone retractions around the midline without a necessary reduction in
growth speed. To examine the relative importance of these two factors, we
determined the frequency of brief retractions of the leader and follower
growth cones within the midline region and outside of it. Ninety percent of
leader axons displayed retraction behavior at the midline; by contrast, only
15% of follower axons showed retraction in this region. When higher retraction
frequency was compensated for, the resulting average absolute growth rate for
leading axons was still significantly lower at the midline (58±9.2
µm/hour) compared with followers (97±5.3 µm/hour)
(Table 1). Outside the midline,
leader and follower growth cones retracted with similar low frequencies and
their average growth rates do not change significantly, even when we account
for these retractions (Table
1).
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Table 1. Longer midline interaction for leader axons is due to decrease in speed
as well as higher frequency of retraction in leader axons
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Leader and follower axons differ in growth cone morphology
Our time-lapse imaging allowed us to resolve entire growth cone shapes
along with a number of filopodia for all leader growth cones and a fraction of
follower growth cones (Fig.
6A-C). To test if growth cones of leader and follower axons differ
in complexity, we computed width/length (w/l) ratios for the two classes of
axons. Leading growth cones (Fig.
6A,B, long pink arrow) were consistently wider and shorter (higher
w/l ratio) than the elongated shape of the follower growth cones
(Fig. 6B,C, long blue arrow).
At the midline, leader growth cone w/l ratios (0.43±0.2, n=12)
were 50% larger than follower growth cone w/l ratios (0.24±0.1,
n=8) (Fig. 6D). The
average growth cone areas for leader and follower axons did not differ
(Fig. 6E), consistent with what
has been found in growth cones of corticothalamic projections in ferrets and
cats (Kim et al., 1991
). In
the midline region, leading axons had up to 50% more filopodia than the
follower axons (average filopodia length did not differ). The filopodia on
leaders were arranged at all angles to the growth cone (see
Fig. 6A, short pink arrows); by
contrast, the filopodia of follower axons were mostly oriented in a forward
direction (Fig. 6A, short blue
arrows). Together, these data show that the growth cones of leader and
follower axons display marked differences as judged by the different w/l
ratios and number and orientation of their filopodia.

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Fig. 6. Growth cone morphology analysis for POC axons. Growth cone morphology can
be visualized during timelapse imaging of the POC axons. (A-C) Representative
growth cones of leader (long pink arrow) and follower (long blue arrow) axons
are shown. Filopodia present on both types of growth cones are also indicated
with smaller arrows (pink for leader and blue for follower). Scale bar: 10
µm. (D) Average growth cone areas do not differ between leader
(n=12) and follower (n=8) axons. Only clearly visible
follower axons were chosen for this analysis. (E) Average width to length
(w/l) ratio plot shows a significantly higher ratio for leader axons
(n=12) compared with follower axons (n=8)
(P<0.05, Student's t-test).
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Direct interaction between leader and follower axons
Because follower axons grew in close contact with the leader axon and had
different midline kinetics, we examined how the presence of the leader axon
affects follower axons in vivo. For this purpose, we examined follower axons
in samples where the leader axon was damaged by intense laser light. Repeated
high laser illumination of single leader growth cones to the point of
saturation causes permanent damage to single axons and has been used as a tool
to ablate cells and their processes (Pike
et al., 1992
; Mayers and Bastiani, 1993). Strong laser
illumination over three to five consecutive time points resulted in leader
axons whose growth cones rounded up and did not recover (n=3;
Fig. 7A). After the growth cone
of the leader was damaged and collapsed in vivo at or before the midline
(Fig. 7A, pink arrow with an
asterisk, see Fig. 7 and Movie 2 at
http://dev.biologists.org/supplemental/),
the nearest follower axon changed its behavior and slowed down within the
midline region (Fig. 7A, blue
arrow with an asterisk), while later follower axons behaved as they normally
would (i.e., crossed the midline swiftly). Thus, early POC axons can adopt
leader-characteristic midline behavior. As long as one axon becomes a leader,
subsequent axons that grow along this tract behave as followers at the
midline.

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Fig. 7. In the absence of a leader axon, follower axons grow more slowly at the
midline. (A) Single time point images showing a leader axon (pink arrow) that
projects towards the midline and is injured right before crossing it (pink
arrow with asterisk). A follower axon behind it (blue arrow) overtakes the
leader axon and crosses the midline (blue arrow with asterisk). After this
axon crosses the midline another axon from the left vrc also grows across the
midline. The two growth cones: the new leader from the right vrc (blue arrow
with asterisk) and the first axon from the left vrc have just undergone their
stereotypic behavior and are not aligned with the POC trajectory until the
last image where their growth cones establish contact with the opposite axon
shaft. (B) Axon distance from midline along the POC trajectory graph showing
three separate cases where follower axon was analyzed after the leader axon
was damaged. The axon graphed in blue corresponds to the follower axon shown
above (blue arrow in A).
|
|
Axon fasciculation can explain the differences between midline
kinetics of leader and follower axons
Leader axons slow down at the midline because they must interpret and
navigate through a complex environment of positive and negative midline cues.
As follower axon growth cones can use leaders as guides to cross the midline,
they are less exposed to midline signals allowing them to cross the midline
swiftly. Thus, a simple difference in growth cones exposure to local cues can
explain the difference between leader and follower axon kinetics at the
midline. Both growth cone morphology differences and the ablation experiments
support this model. To test this hypothesis further, we turned to an
experiment in nature. As mentioned earlier, in 75% of the cases, a leader axon
from one side crosses the and the contralateral first axon grows along the
leader across the midline displaying fast midline kinetics. As leader and
follower growth cone morphology differences correlate with their midline
kinetics, we examined growth cone morphology of these contralateral axons
before and after they encountered the leader axon. If fasciculation of the
growth cone with the leader axon alters its interaction with the environment,
resulting in faster growth at the midline, then we would expect to see a
growth cone morphology change in the contralateral axon accompanying the fast
kinetics we observe in these cases. This change would be expected to occur
even before the growth cone crosses the midline simply because it now grows
along another axon. Following initial contact with a leading axon that has
crossed the midline, growth cones of contralateral axons undergo a drastic
change in shape from complex to elongated even before they themselves cross
the midline (Fig. 8, see Movie
3 at
http://dev.biologists.org/supplemental/).
Thus, simple growth cone shape change brought about by the newly available
opposite commissural axon substrate can switch the midline kinetics of these
axons from leader to follower.

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Fig. 8. Leader axon alters midline kinetics of first contralateral axon at the
midline during commissure formation. Single time point images showing
stereotypic interaction between the initial POC growth cones. Two growth
cones, one from each side, are visible with one already at the midline (left).
Up to this point both growth cones have complex morphologies. Upon contact,
however (middle panel), both growth cones change shape and become elongated
(right) even though the second growth cone has not yet crossed the midline.
When this second axon grows across, it has fast midline kinetics similar to a
follower axon. Scale bar: 10 µm.
|
|
 |
Discussion
|
---|
Here, in vivo microscopy of embryonic zebrafish expressing GFP in the
ventrorostral clusters (vrcs) of cells in the embryonic forebrain permits the
early events involved in establishing the postoptic commissure (POC) to be
followed. The labeled cells in the gata2::GFP fish send their axons along the
POC earlier than any others (22-23 hpf). As the initial axons course towards
the midline, one of them becomes the leader axon, approaching and crossing the
midline first. After a characteristic slowing at the midline, the two leader
axons pass one another and continue on towards their contralateral targets.
Later axons follow the leaders but do not overtake them or behave like them at
the midline. Thus, the leading axons from the vrcs are in the correct place
and show the correct behaviors to serve as the early pioneers of the POC.
Commissural axons have been studied in invertebrates
(Myers and Bastiani, 1993
;
Boyan et al., 1995
) and in
vertebrate spinal cord (Bovolenta and Dodd,
1990
), and appear to share similar mechanisms for building the
early neuronal scaffold. However, little is known about commissural axon
kinetics during early brain development in vertebrates in vivo. Our dynamic
imaging results show behaviors that might not have been expected from
invertebrate studies. First, we find that follower axons can adopt a
pioneering role if the leader is eliminated, contrary to the case in
invertebrates where defined pioneer(s) play a central role in guiding later
axons to their targets (Raper et al.,
1984
; Klose and Bentley,
1989
; Gan and Macagno, 1994;
Hidalgo and Brand, 1997
).
Consistent with these data, removal of some of the primary motoneurons in the
zebrafish spinal cord does not affect the establishment of correct projections
in the remaining population of primary axons
(Pike and Eisen, 1990
). This
suggests that the vrc cells that send their axons along the early POC act as
an equivalence group, with only one or two of the axons serving as leaders.
The signals that promote the leaders or that permit leaders to suppress leader
behavior in followers have yet to be defined. Second, unlike invertebrates,
where bilateral homologues of early commissural axons arrive at the midline
together (Myers and Bastiani,
1993
; Boyan et al.,
1995
), and where cooperative fasciculation between contralateral
homologues of commissural axons appears to be essential for allowing each axon
to cross the midline (Myers and Bastiani,
1993
), we find that this is not always the case in zebrafish POC
formation. In 75% of the timelapses (n=22), an initial axon emerging
from one of the ventral clusters arrived at and crossed the midline before the
contralateral axon got within 15-20 µm of the midline, suggesting that
leader axons can navigate the midline territory alone. Furthermore, although
contralateral Q1 commissural growth cones in grasshopper exhibit strong
affinity for each other at the midline
(Myers and Bastiani, 1993
),
growth cones of leading POC axons do not appear to have equally high affinity
for each other. Contact of leader axon growth cones via their filopodia makes
one or both growth cones jog to the side so that rather than facing each other
they are parallel to each other. As they advance forward, each growth cone
makes contact with the opposite axon shaft directly behind its growth cone,
and then follows it to the other side to establish the initial POC axon
fascicle (data not shown).
Direct interaction between axons has been suggested to influence follower
axon growth direction and growth cone morphologies in retinal ganglion cells
in vitro (Devenport et al.,
1999
). We report a clear behavioral consequence for axons
following an already established track that can be seen in terms of their
growth cone morphology and their midline kinetics in vivo. Similar, growth
cone morphology differences between the initial and later axons have been
noted in invertebrates (LoPresti et al.,
1973
; Raper et al.,
1984
) and in fixed tissues in vertebrate systems
(Kim et al., 1991
; Wilson and
Easter, 1991). We find that axon kinetics and growth cone morphology
correlates with whether the axon is a pioneer or a follower. The fact that
upon elimination of a leader axon, follower axons change their midline
kinetics, and that simple interaction between bilateral leader axons can alter
their growth cone shape and midline kinetics, demonstrates that pioneering
axons and the early follower axons growing along them interact with one
another in vivo. Although it remains possible that these interactions are
indirect, through the leader altering the midline environment, we favor the
interpretation that the difference in kinetics results from direct interaction
between these axons.
One way this might work is that fasciculation between the follower growth
cones and the leader axon simply changes their exposure to the positive and
negative growth signals found at the midline
(Fig. 9). In support of this
model, we observe that only the initial leader axon that pioneers a commissure
displays drastic slowing at the midline and has a complex growth cone. Later
axons following this pioneer do not show the same complex growth cone
morphology, nor do they slow down at the midline. This model predicts that
when the leader is ablated, the substrate it provides for the next axon is
removed, and the next axon becomes more exposed to the environmental cues. As
a result its growth cone morphology as well as its midline kinetics change to
that of a pioneer as observed. Similarly, contralateral leading axons upon
fasciculation change their growth cone morphology and midline kinetics to that
of followers even before crossing the midline as would be expected from this
model. It is also possible that the differences reported here, result from the
balance between positive and negative midline cues that growth cones
interpret, as characterized in vitro in several studies of growth cone
guidance (Song and Poo, 1999
).
In this case, axon fasciculation would speed the kinetics of followers through
the midline milieu, skewing the balance more sharply and resulting in less
retractions and pausing. In either scenario, leader-follower axon
fasciculation ensures that all commissural axons stay sensitive to the midline
cues but permits later axons to cross the midline swiftly and expedite
commissure formation.

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Fig. 9. How fasciculation between commissural axons alters their midline kinetics.
A schematic drawing shows a leader axon (pink) and a number of follower axons
(blue and black) growing through the midline. The leading axon being the
first, is completely exposed to the guidance cues in the environment. Its
growth cone must sense all the positive and negative midline cues and
interpret them accordingly, which results in slow kinetics of leader axons at
the midline where these cues are found. By growing along the leader, follower
axons are less exposed to midline cues. This can happen because their growth
cones are shaped differently, which limits their exposure to conflicting
midline signals and/or because the substrate that the leader axon provides
contributes an extra signal that allows them to grow across the midline
swiftly.
|
|
Studies in invertebrates and vertebrates strongly suggest that midline
cells play an important role in axon guidance
(Hatta, 1992
;
Colamarino and Tessier-Lavigne,
1995
; Greenspoon et al.,
1995
; Matise et al.,
1999
). Mutations affecting midline signaling
(Varga et al., 2001
) or the
presence of midline cells (Pike and Eisen,
1990
; Varga et al.,
2001
) result in axon pathfinding defects. The present focus on the
complex midline domain has assumed that all axons react similarly to these
positive and negative cues, thus slowing down at the midline (Bovalenta and
Dodd, 1990; Mason and Wang,
1997
). Our demonstration that axon kinetics are shaped by an
ongoing interaction between leader and follower axons, in addition to any
midline cues, highlights the importance of investigating the molecular
underpinnings of midline crossing in vertebrates in vivo.
 |
ACKNOWLEDGMENTS
|
---|
We thank Andres Collazo and Olivier Bricaud for suggesting the gata2::GFP
line as candidate for our work as well as Shuo Lin for the gata2::GFP adults.
We thank Kai Zinn, Reinhard Köster, Cyrus Papan and Helen McBride for
discussions and critical reading of the manuscript. This work was supported by
NIH RO1 HD043897.
 |
Footnotes
|
---|
Movies available online
 |
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