Download Use of lipophilic dyes in studies of axonal pathfinding in vivo

MICROSCOPY RESEARCH AND TECHNIQUE 48:25–31 (2000)
Use of Lipophilic Dyes in Studies of Axonal Pathfinding In Vivo
FLORENCE E. PERRIN AND ESTHER T. STOECKLI*
Department of Integrative Biology, University of Basel, CH-4051 Basel, Switzerland
KEY WORDS
growth cone guidance; commissural axons; DiI; tracing
ABSTRACT
Fluorescent lipophilic dyes are an ideal tool to study axonal pathfinding. Because
these dyes do not require active axonal transport for their spreading, they can be used in fixed tissue.
Here, we describe the method we have used to study the molecular mechanisms of commissural axon
pathfinding in the embryonic chicken spinal cord in vivo. Based on in vitro studies, different families
of molecules had been suggested to play a role in the guidance of developing axons. In order to test
their function in vivo, we used the commissural neurons that are located at the dorsolateral border
of the chicken spinal cord as a model system [Stoeckli and Landmesser (1995) Neuron 14:1165–
1179]. Axonin-1, NgCAM, and NrCAM, three members of the immunoglobulin (Ig) superfamily of
cell adhesion molecules (CAMs), were shown to be important for the correct growth pattern of
commissural axons. We studied the effect of perturbations of specific CAM/CAM interactions by
injection of function-blocking antibodies into the central canal of the spinal cord in ovo. After 2 days,
the embryos were sacrificed and fluorescent tracers, such as Fast-DiI, were used to visualize
commissural axons, and thus, to analyze their response to these perturbations in two different types
of fixed preparations: transverse vibratome sections and whole-mount preparations of the spinal
cord. Both pathfinding errors and defasciculation of axons were observed as a result of the
perturbation of CAM/CAM interactions. Microsc. Res. Tech. 48:25–31, 2000. r 2000 Wiley-Liss, Inc.
INTRODUCTION
The correct wiring of the nervous system is a key
issue in developmental neurobiology. During the last
decade a lot of progress has been made towards an
understanding of the molecular mechanisms underlying the growth of individual axons toward their targets.
This progress is on the one hand based on the elucidation of complex interaction patterns of molecules in
vitro, and on the other hand, based on the development
of in vivo assays for testing the role of such molecules in
axonal pathfinding. Outgrowing axons in the developing nervous system are surrounded by a complex
environment that changes over time. Therefore, pathfinding studies in relatively simple in vitro systems can
only mimick isolated aspects of axonal behavior. Alternatively, complicated in vitro systems have to be used
consisting mostly of explant or slice cultures that are
far more difficult to control, but that provide a more
physiological context for growing axons. Ideally, of
course, one sets out to study an axon’s behavior in vivo,
in its natural environment. Common to both the explant cultures and the in vivo experiments is the need
to visualize individual axons in order to monitor their
behavior.
DISCOVERY OF LIPOPHILIC DYES AS A TOOL
FOR AXON TRACING
More than 100 years ago, Santiago Ramón y Cajal
(1995) used the silver staining method developed by
Camillo Golgi to visualize individual neurons. Not only
are his reports of outstanding quality and amazing
detail, but he correctly predicted many of the dynamic
processes involved in the development of the nervous
system, although all was based on these static images.
r 2000 WILEY-LISS, INC.
For instance, he correctly identified the tip of the axons
as the site of growth. He also suggested that axons
could be guided toward their target by chemoattraction,
a mechanism that could be demonstrated only much
later (Lumsden and Davies, 1986; Tessier-Lavigne et
al., 1988). The analysis of the static wiring pattern of
the nervous system was tremendously facilitated by the
use of tracers, substances that are injected, for example, into the target area of axons. They are taken up
by axons and via axonal transport they end up in the
cell somas of projection neurons. One of the most
commonly used substances is horse radish peroxidase
(HRP), which was used as a retrograde tracer starting
in the early seventies. Later, it was shown that under
certain conditions HRP could also be used as an anterograde tracer (Tosney and Landmesser, 1985, 1986).
Because the use of HRP as a tracer depends on an intact
axonal transport, it can only be used in living tissue.
However, the visualization of HRP requires histochemical techniques that are not compatible with living
tissue. Therefore, tissue is commonly fixed after HRP
application and transport, but before processing for
visualization of the tracer. This disadvantage has led to
the search for new tracers with more convenient characteristics (Honig and Hume, 1986, 1989). The carbocyanine dye 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine perchlorate (DiI) was found to fulfill the
requirements very well. It has a high fluorescence
intensity, is very resistant to photobleaching, and is not
*Correspondence to: Esther T. Stoeckli, Department of Integrative Biology,
University of Basel, Rheinsprung 9, CH-4051 Basel, Switzerland. E-mail:
[email protected]
Received 30 April 1999; accepted in revised form 14 July 1999
26
F.E. PERRIN AND E.T. STOECKLI
toxic for living cells (Godement et al., 1987; Honig and
Hume, 1986). Its most important feature, however, is
the fact that it can be used in fixed tissue, as its
distribution is mainly by lateral diffusion in the plasma
membrane rather than by axonal transport. Therefore,
DiI can be used to trace axonal tracts over extremely
long distances in fixed tissue that can be kept for weeks
to allow sufficient time for the diffusion of the dye
(Godement et al., 1987). Today, various derivatives of
DiI have been synthesized. They differ in their diffusion
rate and their fluorescence characteristics. As dyes are
available in several colors, different neuronal populations can be labeled individually and their trajectories
can be traced in the same slice or tissue. An impressive
application of this was the use of lipophilic dyes in an
automated analysis of a large screen for pathfinding
mutants in the retinotectal system of zebrafish (Baier
et al., 1996). Due to its non-toxicity and high fluorescence intensity, DiI is well suited for the tracing of
extending axons in vivo (for a review see Fishell et al.,
1995). Taking advantage of the transparency of zebrafish embryos Pike and Eisen (1990) demonstrated
that individual motor axons could be followed as they
navigated toward their target muscles. Kaethner and
Stuermer (1992) used time lapse video microscopy to
follow in vivo the terminal arbor formation of individual
retinal ganglion cell axons in the tectum.
Carbocyanine dyes have also been used successfully
for time-lapse recording of axonal behavior in higher
vertebrates. However, as brain tissue from mammalian
or chicken origin is not transparent, axonal tracing is
more difficult and requires sectioning (Dailey et al.,
1994). With the development of confocal and twophoton microscopy, the depth in which labeled axons
can be followed has increased (Piston, 1999).
COMMISSURAL AXONS OF THE SPINAL
CORD AS A MODEL SYSTEM TO
STUDY MOLECULAR MECHANISMS
OF AXON GUIDANCE
During the development of the nervous system, axons
have to navigate through the preexisting tissue and to
establish correct connections with their targets. For
this purpose, the axon has a highly motile structure at
its tip that acts as a sensor for guidance cues presented
by the environment (Vogt et al., 1996). The growth cone
guides the axon along its trajectory by constantly
sampling and integrating the molecular signals derived
from the interaction of its surface molecules with such
guidance cues. The result of this process is translated
into an intracellular signal that is transported back to
the nucleus where it elicits the neuron’s response. We
still have limited knowledge about the crosstalk of
surface molecules with the cytoskeleton, the effector of
the axon’s growth behavior. However, depending on the
nature of the incoming signal the axon will continue to
grow, stop, turn, or retract. Based on in vitro studies, a
complex interaction pattern of surface molecules has
been elucidated. Different families of molecules have
been identified as candidates for molecular cues involved in the guidance of growing axons (Goodman,
1996; Tessier-Lavigne and Goodman, 1996; VarelaEchavarrı́a and Guthrie, 1997). Based on their effect,
they are divided into two groups, chemorepulsive and
chemoattractive guidance cues (Tessier-Lavigne and
Goodman, 1996). Both groups can be further subdivided into short-range and long-range guidance cues.
By definition, short-range cues are locally active,
whereas long-range cues can act over some distance,
because they are secreted and to some extent diffusible.
A particular molecule can have different, even opposite
effects on distinct neuronal populations. For example,
netrin-1 was first identified as a chemoattractive molecule for commissural axons (Kennedy et al., 1994;
Leonardo et al., 1997; Serafini et al., 1994), and subsequently, it was demonstrated to be a chemorepellent for
trochlear motor axons (Colamarino and Tessier-Lavigne, 1995). As both some short- and long-range guidance cues have been identified, the commissural axons
of the developing spinal cord are one of the best
characterized model systems for pathfinding at the
molecular level (for reviews see Stoeckli, 1998; Stoeckli
and Landmesser, 1998). Commissural axon pathfinding
has been studied at the molecular level in Drosophila
(Kidd et al., 1998, 1999; Seeger et al., 1993), in Caenorhabditis elegans (Zallen et al., 1998), and in chick
(Stoeckli and Landmesser, 1995; Stoeckli, 1997).
In chick, at the lumbosacral level, commissural neurons located in the dorsal half of the embryonic chicken
spinal cord, close to the dorsal root entry zone, start to
extend axons at stage 19 (Hamburger and Hamilton,
1951). The first fibers reach the floor plate at about
stage 21. After crossing the midline, they turn rostrally
along the contralateral border of the floor plate. TessierLavigne and colleagues showed that floor-plate explants co-cultured with commissural neurons in a
three-dimensional collagen matrix had a chemoattractive effect on commissural axons (Tessier-Lavigne et al.,
1988). Subsequently, this in vitro assay system was
used to purify the chemoattractive activity derived
from the floor plate. Netrin-1, a 78 kDa protein expressed and secreted by the floor plate, was the first
long-range guidance cue identified at the molecular
level (Kennedy et al., 1994; Serafini et al., 1994).
Based on their complex interaction pattern in vitro
and based on their neurite outgrowth-promoting activity, cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily were suggested to play a role in
axonal pathfinding (Brummendorf and Rathjen, 1996;
Burden-Gulley and Lemmon, 1995; Doherty and Walsh,
1994). Such a role was corroborated, for instance, by in
vivo studies in chicken embryos (Stoeckli and Landmesser, 1995; Tang et al., 1992, 1994).
In this paper, we will focus on the functional studies
done in chick and describe the use of carbocyanine dyes
as axonal tracers in an in vivo pathfinding study, where
FastDiI was used to trace commissural axons in the
embryonic chicken spinal cord after perturbation of
various interactions between cell adhesion molecules
(Stoeckli and Landmesser, 1995; Stoeckli et al., 1997).
MATERIALS AND METHODS
In Vivo Injections
Fertilized eggs were obtained from a local supplier.
They were incubated at 38.5–39°C, before they were
opened on the third day of incubation for in ovo
injections. To have access to the embryo, a window was
cut into the eggshell. In order to facilitate repeated
USE OF DII IN AXONAL PATHFINDING STUDIES
opening and closing, we sealed the window with a
coverslip using melted paraffin. Proper sealing was
crucial to avoid dehydration of the embryo. Chicken
embryos were staged according to Hamburger and
Hamilton (1951) at the time of injection. For injections
into the spinal cord, the extraembryonic membranes
were cut with spring scissors and removed from the
lumbosacral region. The injections were made just
rostral of the hindlimb, directly into the central canal of
the spinal cord, using glass electrodes attached to a
piece of teflon tubing. We controlled the injection volume by mouth and did not use a mechanical device. The
electrodes were pulled on a Narishige PC-10 electrode
puller to a tip diameter of 5 µm. In order to control the
localization of the injection site, the quality of the
injections, and the injected volume, 0.04 % Trypan Blue
was added to the injected solutions. In order to assure
the presence of function-blocking antibodies throughout the period of axonal pathfinding in sufficient quantity, high concentrations had to be injected to make up
for the dilution due to diffusion and developmentdependent increase in volume of the spinal cord. The
injections were repeated every 8 hours. Thus, embryos
were injected 4 to 5 times between stages 18 and 24.
Because the injectable volume was very small (between
0.05 and 0.1 µl for early stages), the antibody concentrations used were 10 mg/ml or higher. The anti-axonin-1
antibody used was raised in goat as described in Ruegg
et al. (1989); the rabbit antibody against NgCAM was
described previously in Landmesser et al. (1988). The
antibodies against NrCAM were raised in rabbit against
affinity-purified NrCAM as described by de la Rosa et
al. (1990).
The embryos were sacrificed and dissected between
stages 25 and 26. After stage 26, the associational
neurons that are localized close to the commissural
neurons would have confused the results, as they
project their axons ipsilaterally. Embryos injected with
nonimmune serum were not different from non-injected
embryos with respect to both development and commissural axon pathfinding. The pathfinding behavior of
commissural axons was compared in two different types
of spinal cord preparations. In transverse vibratome
sections, the ipsilateral growth pattern was compared
between control and experimental embryos. The ipsilateral turns and the defasciculation along the contralateral border were more obvious in whole-mount preparations, the so-called open-book preparations. For
transverse vibratome sections, embryos were decapitated, dissected, and pinned down in a Sylgard dish for
fixation in 4% paraformaldehyde in PBS for at least 3
hours at room temperature or at 4°C overnight. The
embryos were rinsed in PBS and embedded in 7.5%
ultra low-melting agarose (Sigma, Buchs, Switzerland).
The blocks were trimmed and mounted such that
transverse sections of 250 µm could be cut with an
OTS-3000–04 oscillating tissue slicer (Electron Microscopy Sciences, Fort Washington, PA). For open-book
preparations, the spinal cord was removed from the
embryo after a ventral laminectomy using tungsten
needles. The roof plate was cut and the cord was flipped
open, flattened, and pinned down in a Sylgard dish for
27
fixation. Fixation was as described for transverse sections.
Dye Injections
We used mainly FastDiI (1,18-dilinoleyl-3,3,38,38tetramethylindocarbocyanine 4-chlorobenzenesulfonate) obtained from Molecular Probes, Eugene, OR,
dissolved at 5 mg/ml in methanol. The dye was injected
with glass electrodes pulled to a tip diameter of up to 3
µm into the area of the somas of commissural neurons
in either tissue slices or whole-mount preparations.
After incubation for 36–48 hours, the preparations
were mounted in PBS between coverslips and viewed
with conventional fluorescence or confocal microscopy.
As the fluorescence intensity of the lipophilic dyes is
high, but the thickness of the preparations is rather big,
objectives with a large focal depth may sometimes give
better results than objectives with higher numerical
aperture, but small focal depth.
RESULTS
Use of Fluorescent Tracers in the Analysis of the
Role of Guidance Molecules in Commissural
Growth Cone Guidance and Fasciculation
The Ig superfamily CAMs, axonin-1 and NgCAM, are
expressed on commissural axons during initial stages of
axon extension (Shiga et al., 1990; Shiga and Oppenheim, 1991; Stoeckli and Landmesser, 1995), whereas
NrCAM is expressed on floor-plate cells (Krushel et al.,
1993; Stoeckli and Landmesser, 1995). In vitro, axonin-1 was shown to bind to NrCAM (Suter et al., 1995),
and furthermore, axonin-1 was shown to interact with
NgCAM in the plane of the same membrane (Buchstaller et al., 1996; Stoeckli et al., 1996; Sonderegger et
al., 1998). To test for a role of these molecules in
commissural axon guidance in vivo, perturbation experiments were carried out (Stoeckli and Landmesser,
1995; Stoeckli, 1997). Function-blocking antibodies
against axonin-1, NgCAM, or NrCAM were injected
into the central canal of the developing chicken spinal
cord in ovo (Fig. 1A) (see Materials and Methods for
details). These injections were repeated during the time
window of commissural axon pathfinding. Between
stages 25 and 26, after commissural axons had turned
into the longitudinal axis to extend along the contralateral floor-plate border, the embryos were sacrificed and
fixed for analysis. Making use of the capability of
fluorescent tracers to diffuse in fixed tissue, we visualized the trajectories of commissural axons in control
and experimental embryos. Axonal trajectories in two
different types of spinal cord preparations were analyzed and compared. Transverse vibratome sections
(250 µm) of the spinal cord were cut from the lumbosacral level to look at the ipsilateral pathfinding behavior
of commissural axons (Fig. 1B). To assess pathfinding
errors and the growth pattern of commissural axons in
the longitudinal axis along the contralateral side of the
floor plate, the ‘‘open-book’’ preparation was more useful. This whole-mount preparation was obtained by
flattening the spinal cord after transection of the roof
plate (Fig. 1C). Lipophilic dyes were then injected in the
area of the cell bodies to visualize commissural axon
trajectories.
28
F.E. PERRIN AND E.T. STOECKLI
Fig. 1. In vivo perturbations and analysis of commissural axon
pathfinding. The role of specific guidance cues in the pathfinding of
commissural axons in the embryonic chicken spinal cord in vivo was
tested by perturbation experiments in ovo. For that purpose, functionblocking antibodies were injected directly into the central canal of the
developing spinal cord in ovo (A). The injections were repeated
throughout the time window of commissural axon pathfinding (stages
18 to 24). Between stages 25 and 26 (E 4.5), the embryos were
sacrificed and the trajectories of commissural axons of the lumbosacral
level of the spinal cord were analyzed either in transverse vibratome
sections (B) or in whole-mount preparations (open-book preparations,
see Materials and Methods for details; C). To visualize the axonal
trajectories FastDiI dissolved in methanol was injected into the area of
the cell bodies of commissural neurons (marked with an asterisk in B
and C). In control embryos, commissural axons project their axons
ventromedially toward the floor plate. Axons cross the midline and
turn into the longitudinal axis along the contralateral border of the
floor plate (FP).
In non-injected and control-injected embryos, commissural axons grew as a discrete bundle toward the floor
plate (Fig. 2 A–C; see Stoeckli and Landmesser, 1995,
for more details). Similarly, in open-book preparations
the DiI-labeled axons were found to cross the floor plate
and to turn rostrally along the contralateral border of
the floor plate in a fasciculated manner (Fig. 2C). After
perturbation of axonin-1 interactions by injection of
function-blocking anti-axonin-1 antibodies, axons grew
in a defasciculated manner both before and after crossing the floor plate (Fig. 2 D, compare the distance
between arrowheads in control [Fig. 2A] and experimental embryos [Fig. 2D]). Even more strikingly, some
axons committed pathfinding errors (arrows in Fig. 2E
and F). They failed to cross the midline and joined the
longitudinal tract along the ipsilateral border of the
floor plate (Fig. 2F, arrow). Using the same approach, a
defasciculation of commissural axons was also shown to
result from the perturbation of NgCAM interactions.
However, in contrast to anti-axonin-1 injected embryos,
no pathfinding errors were found in anti-NgCAMtreated embryos. The perturbation of NrCAM interactions resulted in similar pathfinding errors as the ones
found after perturbation of axonin-1 interactions. Together with the notion that axonin-1 and NrCAM were
heterophilic binding partners in vitro (Suter et al.,
1995), these findings strongly suggested that NrCAM
and axonin-1 were binding partners in the guidance of
commissural axons across the floor plate (Stoeckli and
Landmesser, 1995; Stoeckli et al., 1997). Axonin-1 and
NgCAM seemed to be responsible for the fasciculation
of commissural axons both before and after crossing the
floor plate. Thus, it was concluded that NgCAM was
involved in the fasciculation of commissural axons but
not in their pathfinding. Taken together, these results
showed that the defasciculation of commissural axons
was not responsible for their failure to cross the floor
plate, rather that these axons were capable of reading
the guidance cues presented by the floor-plate surface
on an individual basis.
USE OF DII IN AXONAL PATHFINDING STUDIES
29
Fig. 2. In the absence of axonin-1 interactions commissural axons
commit pathfinding errors. Specific interactions between the candidate guidance cue axonin-1 and its binding partners were perturbed
by the injection of function-blocking antibodies into the spinal cord of
chicken embryos in ovo. The effect of these perturbations on commissural axon trajectories were analyzed in transverse sections (A, B, D,
E) and whole-mount preparations (C, F) of the spinal cord. FastDiI
was injected into the area of commissural neuron somas to trace their
axon trajectories (see Fig.1). In control embryos (A–C), commissural
axons project ventromedially toward the floor plate as a compact
bundle (A and B; the bundle is outlined with arrowheads in A). All
axons cross the midline and turn rostrally along the contralateral
border of the floor plate (C). In contrast, in embryos treated with
anti-axonin-1 antibodies (D– F), commissural axons grow defasciculated (D and E, compare width of the bundle in A and D). In the
absence of axonin-1 interactions, some commissural axons commit
pathfinding errors (marked with arrows in D through F). Instead of
crossing the midline, they turn prematurely along the ipsilateral
border of the floor plate (F). Arrowheads in A and D indicate the width
of the commissural axon trajectory toward the floor plate. Arrows
indicate axons committing pathfinding errors (D– F). Bar ⫽ 100 µm in
A and D, 50 µm in B and E, and 40 µm in C and F.
DISCUSSION
The advantage of the method used for this pathfinding study is the possibility to follow the behavior of
commissural axons in their natural environment. The
perturbation of a specific guidance cue thus affects the
interactions of this particular molecule with all its
binding partners, whereas independent molecular interactions are left untouched. Although the intact spinal
cord of a chicken embryo does not allow the optical
access to follow living axons directly, the fact that the
trajectory of commissural axons can be visualized after
fixation provides a means to circumvent this problem
and to dissect the role of individual guidance cues. We
have successfully used this approach to study the role of
CAMs (described here) and floor-plate derived extracellular matrix molecules (Burstyn-Cohen et al., 1999).
An advantage of the visualization of axons by dye
injections rather than by immunohistochemistry is the
independence from a particular marker protein. Even if
such markers exist, their expression could be influenced by the perturbation of cell/cell contacts as a result
of the absence of specific CAM/CAM interactions, and,
thus, the analysis could be compromised. Because
lipophilic dyes spread by diffusion in the entire cell
membrane, axon tracing with FastDiI allows the visualization of fine structural details, such as filopodia on
growth cones (Fig. 3). In a recently published study,
Liljelund and Levine (1998) compared morphology and
behavior of DiI-labeled growth cones in living slices
with fixed preparations of early postnatal rat cerebellum. Similarly, lipophilic dyes were used in the retinotectal system to correlate growth cone morphology with
behavior at specific choice points (Godement et al.,
1994; Mason and Wang, 1997). All these studies would
not have been possible with the use of HRP or similar
tracers that require tissue processing for their visualization.
Because labeling of axonal tracts with lipophilic dyes
is compatible with subsequent immunohistochemistry,
a combination of dye injections and staining with
fluorescent antibodies is possible (Elberger and Honig,
1990). Thus, lipophilic dyes are an invaluable tool for
many different types of studies. They have been used
successfully in pathfinding studies in many species,
30
F.E. PERRIN AND E.T. STOECKLI
Fig. 3. Lipophilic dyes can be used to visualize structural details of
growth cones in tissue slices. Lipophilic dyes spread over the entire
cell membrane by lateral diffusion. Because there is no leakage from
cell to cell, structural details of individual growth cones can be
resolved in tissue slices or whole-mount preparations after application
of FastDiI to the cell body.
both in living as well as fixed preparations. Their
versatility will certainly make them the tracer of choice
for many future applications.
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