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New Ways to Look at Axons in Caenorhabditis elegans
Max-Planck-Institut für medizinische Forschung, Jahnstr. 29, 69120 Heidelberg, Germany
nervous system; GFP; confocal microscopy
In the nematode Caenorhabditis elegans, a well-established model organism for the
analysis of nervous system development and function, nerve processes can be labelled in the intact
animal with markers based on the ‘‘Green Fluorescent Protein’’ (GFP). The generation of GFP
variants with improved brightness and modified emission spectra potentiated the use of this marker
for in vivo labelling of subcellular structures. This made it possible to label different groups of
neurons and their axons in the same animal with GFP variants of different spectral characteristics.
Here I show with double labelling experiments that spatial relationships of axons in small axon
bundles can now be resolved at the light microscopic level. In the future this will largely circumvent
the need for time-consuming electron microscopic reconstructions to detect local defects in axon
outgrowth. Furthermore, I demonstrate that neuronal processes can now be traced even in the head
ganglia, an area of the nervous system that was previously almost inaccessible for analysis due to
the compact arrangement of cell bodies and axons. Microsc. Res. Tech. 48:47–54, 2000. r 2000 Wiley-Liss, Inc.
Some 30 years ago Sydney Brenner and his colleagues started to cultivate a small free-living soil
nematode called Caenorhabditis elegans in their lab. A
major goal was to use this animal to study development
and function of the nervous system, specifically how
these processes are controlled genetically. All developmental stages of C. elegans are transparent. This
enabled Sulston and co-workers to follow cell divisions
and migrations in the intact developing animal. They
described the complete developmental history and final
position of every cell in the animal (Sulston et al., 1983;
Sulston and Horvitz, 1977). These positions are highly
invariant from animal to animal and can be used by the
experienced researcher to identify a particular cell.
This knowledge allowed White and co-workers to initiate an even more ambitious project, the complete
electron microscopic (EM) reconstruction of the nervous
system including the connections among neurons. In
serial sections they traced all neuronal processes back
to their cell bodies in order to determine their identity.
Chemical and electrical synapses were scored and
revealed how the nervous system is wired (White et al.,
1986). Almost all of the neurons have a very simple
morphology with only one or two processes that typically are unbranched. Synaptic connections were mainly
found in process bundles, where an axon develops a
local swelling and establishes a synapse with a neighbouring dendrite. Therefore, a particular process is
always found in a defined position within a process
bundle surrounded by its synaptic targets. Consequently, every bundle is a highly ordered structure
where the relative position of axons reflects the connectivity (Fig. 1). Outgrowing processes in C. elegans find
their targets one by one on their way as they grow out,
and they have to navigate very precisely over the entire
distance in order not to miss any of them.
In their initial attempt to identify and analyse the
genes that control neuronal development and function,
Brenner colleagues isolated a number of neurological
mutants (Brenner, 1974). These were first identified as
having characteristic behavioural defects, e.g., defects
in the coordination of their movement. EM reconstructions revealed specific defects in axonal outgrowth for
some of them that could be interpreted in terms of
specific guidance signals affected or cell identities misspecified. It is important to note that the extremely
high degree of invariance in the anatomy of C. elegans
allowed researchers already early on to analyse in
detail the consequences of experimental manipulation
without the need for molecular markers that became
available only later. However, the number of neurological mutants that were isolated soon exceeded the
number that could be analysed by EM reconstructions.
Histochemical markers were discovered that allowed
visualisation of groups of neurons with a common
neurotransmitter (McIntire et al., 1993) or sensory
neurons with openings to the outside that could be filled
with fluorescent dyes simply by soaking the animal in
dye solution (Hedgecock et al., 1985). These staining
methods were used to rapidly screen the existing
mutant collection in order to identify those mutants
that had axonal outgrowth defects (Hedgecock et al.,
1985; McIntire et al., 1992; Siddiqui and Culotti, 1991).
One disadvantage of histochemical markers compared to EM reconstruction is the limited spatial
resolution. The need to kill and fix the animal before
the staining procedure frequently reduces the resolution even further compared to observations of the intact
tissue in a living animal, so that it becomes difficult or
even impossible to discern certain details of axon
trajectories. The introduction of the ‘‘Green Fluorescent
Protein’’ (GFP) as a marker for in vivo labelling (Chalfie
et al., 1994) overcame some of these limitations. Cur*Correspondence to: Harald Hutter, Max-Planck-Institut für medizinische
Forschung, Jahnstr. 29, 69120 Heidelberg, Germany. E-mail: hutter@
Received 31 May 1999; accepted in revised form10 August 1999
Fig. 1. The nervous system of C. elegans. A: A schematic drawing of
the head region of C. elegans illustrating the location of various parts
of the nervous system. Interneuron cell bodies are located in head
ganglia close to the nerve ring, whereas motorneuron cell bodies are
positioned along the ventral midline. Note that interneuron axons use
different routes into the nerve ring and run in defined positions within
the ring. B: Cross-section through the ventral cord after White et al.
(1986). The ventral cord consists of two longitudinal axon tracts
separated by a hypodermal ridge. Also positioned between the axon
tracts are cell bodies of motorneurons. Most of the axons run in the
right tract. The spatial relationships of axons within the bundle are
highly invariant from animal to animal. Interneuron axons (AVB,
AVD, AVE , AVA, PVC) and motorneuron axons form distinct subbundles and maintain a particular order even within the subbundle.
C: Synaptic connections between interneurons and motorneurons
(type A and type B) within the ventral cord. A large number of synaptic
connections is formed among the interneurons and between interneurons and motorneurons. Arrows point from the presynaptic to the
postsynaptic process. Since synapses are formed only between processes that contact each other, the connectivity is reflected in the
relative positioning of neuronal processes.
rently several GFP variants with improved brightness
and different colours are available (Heim and Tsien,
1996; Ormö et al., 1996).
Here I demonstrate that the use of GFP in combination with advanced microscopy and imaging techniques
allows a new view of the nervous system of C. elegans.
Double labelling experiments reveal that the spatial
resolution in an intact animal is sufficient to visualise
relative positions of axons even within small axon
bundles. In the brain region, which was virtually
inaccessible at the light microscopic level due to its
complexity of cell bodies and neuronal processes, GFP
labelling now allows the detailed reconstruction of axon
GFP Constructs
The following plasmids were used as a starting point
for cloning experiments. KP#6 kindly provided by J.
Kaplan contains the glr-1 promoter for expression of
reporter genes in a subset of interneurons (Hart et al.,
1995). pPD95.75 kindly provided by A. Fire is a promoterless vector containing the S65C variant of GFP (green
GFP). pPD115.46 also provided by A. Fire is a promoterless vector containing the Y66W N146I M154T V163A
variant of GFP (cyan variant or CFP). The 5.3 kb
PstI-BamHI fragment from KP#6 containing the glr-1
promoter was cloned into pPD95.75 resulting in
pG75#KP6 and also into pPD115.46 resulting in
Transgenic Strains
Transgenic strains were made using standard techniques (Mello and Fire, 1995). Briefly, plasmid DNA
(pG75#KP6 or pG46#KP6) was injected into C. elegans
hermaphrodites. dpy-20(⫹) DNA was used as coinjection marker; injections were made into a dpy20(e1282ts) strain. Stables lines were generated by
gamma-irradiation followed by outcrossing. Animals
where motorneurons are labelled with GFP and interneurons with CFP were generated by crossing a strain
carrying pG46#KP6 with one expressing GFP in DA
and DB motorneurons (kindly provided by J. Culotti).
Microscopy and Imaging
Images for Figure 2 were taken on a Leica SP confocal
system equipped with an Argon laser using a Plan Apo
100x, 1.4NA oil objective. CFP was excited at 458 nm,
GFP at 488 nm. Emitted light was collected between
468 and 482 nm for CFP using a 465-nm dichroic filter,
and between 500 and 560 nm for GFP using a 488-nm
dichroic filter. Sequential scans were used to collect
images in the two channels.
Images for Figure 3 were taken on a Leica NT
confocal microscope equipped with an Argon-Krypton
laser using a Plan Apo 63x, 1.4NA oil objective. GFP
was excited at 488 nm, a TK500 dichroic filter was used
together with a BP525/50 barrier filter.
Images were further processed with the Huygens2
(Bitplane AG, Switzerland) deconvolution software and
displayed using the Imaris program. Within Huygens2
the Maximum Likelihood Estimation method (MLE)
algorithm (Snyder et al., 1992) was used for deconvolution with a calculated point spread function.
Determining the Relative Positions of Axons
Within an Axon Bundle
In C. elegans, the relative position of a particular
axon within an axon bundle is well conserved. Determining these positions so far was only possible in timeconsuming EM reconstructions. Using GFP as a marker
in living animals, I attempted to visualise these spatial
relationships at the light microscopic level. To this end,
I generated transgenic animals expressing two variants of GFP in different groups of axons. A cyan variant
(CFP) was used to label interneurons of the motor
circuit whereas a green variant was used to label a
subset of motorneurons. Both groups of axons are found
in the major longitudinal axon tract, the ventral cord.
From EM reconstructions we know that interneurons
and motorneurons form distinct subbundles within the
ventral cord with the motorneuron bundle lying ventrally adjacent to the interneuron bundle (White et al.,
1976; Fig. 1B). This relative position is maintained over
a long distance and reflects the local synaptic connections within the ventral cord (Fig. 1C). The entire
ventral cord in cross-section is about 2 µm wide with
individual axons about 0.2 µm in diameter. A single
axon, therefore, is at the resolution limits of a light
microscope and with current histochemical markers
the relative positions of axons within an axon bundle
cannot be resolved. How far can the resolution limits be
pushed using modern microscopy and imaging techniques together with in vivo markers that allow imaging of an intact animal?
3D image stacks of the ventral cord of double-labelled
animals were recorded with a confocal microscope.
Figure 2A shows a central focal plane with the interneuron bundle in green and the motorneuron bundle in red.
The relative positions of these axons clearly can be
determined. The motorneuron axons are positioned
ventrally adjacent to the interneuron subbundle (Fig.
2B). Both groups of axons are tightly bundled together.
To further improve the spatial resolution and quality
of the image a deconvolution algorithm was applied
(Maximum Likelihood Estimation method (MLE) algorithm (Snyder et al., 1992)). The resulting pictures (Fig.
2C, D) demonstrate that this indeed leads to a sharper
image with less blur around the edges of axons, which
makes it easier to determine the borders of the subbundles.
CFP and GFP have overlapping excitation and emission spectra and it is impossible to completely separate
the two variants in standard fluorescence microscopes
equipped with a mercury lamp for illumination. On a
Leica SP confocal microscope, however, the emission
window can be adjusted freely, a feature that allows one
to determine for each sample individually the windows
that minimise bleed-through (in this example CFP was
detected between 465 and 482 nm, GFP was detected
between 500 and 560 nm). Laser strength for illumination and photomultiplier settings for detection can be
adjusted so that bleed-through is essentially undetectable (Fig. 2E–H). This allows a clean separation of CFP
and GFP signals.
Interneuron and motorneuron axons maintain their
relative positions over the entire length of the ventral
cord. Figure 2I shows a larger region of the ventral cord
between two neighbouring motor neuron cell bodies.
Again the motorneuron axons can be seen running
adjacent to the interneuron axons. A single axon that
clearly runs apart from the interneuron bundle on the
dorsal side of the ventral cord (small arrow) belongs to
the AVG neuron. This is the first axon to grow into the
ventral cord on the basement membrane that separates
muscle and epidermis. Figure 2J–M show crosssections at 4 different positions indicated with arrowheads in Figure 2I. Note that the motorneurons are
always found in the same position with respect to the
interneurons. Note also that the single AVG axon which
runs only about two axon diameters away from the
interneuron bundle can easily be resolved in the crosssections (arrows in Fig. 2J,K).
Axon Trajectories in the Brain Region
The major neuropil in C. elegans is called the nerve
ring. This is a large bundle of about a hundred axons
that loop around the pharynx (Fig.1A). The majority of
synapses is found here. The nerve ring is surrounded by
Fig. 2.
a number of ganglia containing cell bodies of neurons
projecting their axons into the ring. There are a number
of different entry and exit points and a major nerve
leaving the ring on the ventral side, which eventually
becomes the ventral cord (Fig. 1). The large number of
cell bodies, the short distances between cell bodies and
nerve ring, and the compact architecture of the ring
itself made it almost impossible to discern individual
features with histochemical markers. The use of GFP
again changes this situation and allows one to trace
axon trajectories in the brain area even in a complex
staining pattern with many axons labelled.
GFP expressed under the control of a glutamate
receptor promoter (glr-1 promoter) was used to visualise axons in the brain region. The cells expressing glr-1
have been described (Hart et al., 1995) and their axon
trajectories are known from EM reconstructions (White
et al., 1986). In Figure 3 a number of axon trajectories
into the nerve ring are illustrated. Figure 3A shows a
side view of the brain area. The picture is a projection of
several focal planes. Starting from the cell body, the
AVJL axon can be followed as it projects anteriorly
towards the nerve ring. Upon entering the nerve ring
subdorsally, it abruptly turns and runs in a posterior
position within the ring towards the dorsal midline
where it crosses over to the contralateral side. Individual focal planes illustrating different parts of the
trajectory are shown in Figure 3B–D. A dorsal aspect of
the nerve ring illustrating the same trajectory is displayed in Figure 3E.
Another neuron, RIML, whose cell body is close by,
uses an entirely different route into the nerve ring. It
first projects ventrally through the amphid commisure
into the ventral cord, where it turns anteriorly to enter
the nerve ring from the ventral side (Fig. 3F).
Even for neurons that have their bodies immediately
adjacent to the nerve ring, it is possible to follow their
axons into the ring. In Figure 3G, the RMDL axon is
shown entering the nerve ring laterally and extending
into the anterior part of the nerve ring. Upon entering a
process bundle, axons typically maintain their relative
positions for long distances (see Fig. 2), i.e., they travel
in a constant neighbourhood surrounded by the same
axons, which frequently are synaptic partners. Many
processes, however, make abrupt changes of neighbourhood at certain points. These points frequently are
found where process bundles meet or split up (White et
al., 1986). Within the nerve ring, a prominent point of
change is the dorsal midline. Certain processes terminate here whereas others make abrupt changes in
direction as they cross the midline. In Figure 3H, axons
can be seen as they switch from a posterior position
Fig. 2. Double labelling of axons in the ventral cord. A: False colour
image of interneuron (CFP-labelled) and motorneuron axons (GFPlabelled) in the ventral cord. Interneurons axons are in green,
motorneuron axon in red. Side view. Animals are oriented with
anterior to the left, dorsal is up. Scale bar ⫽ 2 µm. B: Cross-section
through the ventral cord at the position marked by the arrowhead in A.
C,D: Same images after deconvolution with Huygens2. E–H: Single
channel images of C,D. I: A larger region of the ventral cord with cell
bodies of motorneurons visible. The single AVG axon which is the
dorsalmost axon in the ventral cord can be seen running separate from
the interneuron bundle (small arrow). J–M: Four cross-sections
through the ventral cord at positions indicated by arrowheads in I.
within the ring to a more anterior one after crossing the
dorsal midline. The high spatial resolution achieved by
the use of GFP markers in living animals in combination with confocal microscopy allows the analysis of
axonal outgrowth in areas that were almost inaccessible before.
Understanding the underlying logic of a complex
process like the development of the nervous system
during embryogenesis first of all requires a thorough
description of the fully developed structure and the
intermediates stages through which it develops. To this
end, appropriate methods of visualisation are essential,
for the developing nervous system most importantly a
method to visualise nerve processes at high resolution.
For the nematode C. elegans, a uniquely detailed
description of the adult nervous system is available in
the form of EM reconstructions (White et al., 1986).
This information serves as a basis to interpret the
results of various experimental manipulations, most
notably the inactivation of individual genes that play a
role in various aspects of neuronal development. EM
reconstructions were used to analyse the defects in the
first neurological mutants isolated by Brenner and
colleagues (Brenner, 1974). However, since they are
very time-consuming and require extraordinary experience, they were the method of choice only for a small
number of researchers and a few selected experiments.
Many researchers had to limit themselves to an analysis at the light microscopic level with its reduced spatial
resolution. This imposed certain constraints on the
analysis especially in particular regions of the nervous
system like the brain with its complex and compact
architecture. As a consequence, certain aspects of neuronal development like guidance of axons in local
environments or synaptic target recognition were difficult to analyse.
The introduction of the Green Fluorescent Protein
(GFP) as in vivo marker was a technical revolution for
cell biologists. In C. elegans, with its small size and
transparent body, GFP tagging can be used to follow
developmental processes in the intact animal with high
temporal and spatial resolution.
Microscopy and imaging techniques have also been
improved significantly over the last years. The introduction of confocal microscopy techniques together with
improved image processing algorithms enable us now
to push the resolution limits of the light microscope.
New Avenues for the Analysis of Neuronal
Development in C. elegans
Distances within the nervous system of C. elegans are
small enough, so that axons do not have to branch
extensively in their target area to contact different
synaptic partners. Axons develop local swellings and
establish synapses with neighbouring processes within
process bundles (White et al., 1986). As a consequence,
these bundles are highly ordered structures where
individual axons are found in fairly invariant positions
within the bundle. The local signals that are used by
axons to navigate within an axon bundle as well as the
signals that initiate synapse formation with particular
neighbours are essentially unknown. Identifying these
signals was difficult, since the only way to determine
Fig. 3.
the position of a particular axon within a process
bundle were EM reconstructions. The results presented
here illustrate that it is now feasible to determine the
relative position of axons within a process bundle at the
light microscopic level, facilitating future studies of
local guidance signals in C. elegans.
A few signals that provide long-range orientation
cues for axons have been identified already. One important signal for guidance in dorsoventral directions is
encoded by the gene unc-6 (Ishii et al., 1992). The unc-6
gene product, netrin, is thought to form a gradient
along the along the dorsoventral axis with its highest
concentration on the ventral side (Wadsworth et al.,
1996). Certain axons are attracted towards the ventral
midline by netrin while others are repelled (Hedgecock
et al., 1990). Analysis of the expression pattern revealed that netrin is also expressed by cells bordering
the nerve ring (Wadsworth et al., 1996). This implies
that unc-6 signalling might also be important in the
developing nerve ring. Axon trajectories, however, are
difficult to determine in this region of the nervous
system with histochemical markers due to the compact
architecture of the head ganglia and process bundles.
GFP markers in combination with confocal microscopy
and imaging techniques now provide access to these
regions of the nervous system of C. elegans. An evaluation of outgrowth defects of axons in this area is
possible now and will lead to a deeper understanding of
how the wiring of the nervous system is controlled.
Potential and Limitations of GFP Markers
An individual GFP-labelled axon can be followed
until it joins a bundle containing equally labelled
axons. Beyond this point, it is impossible to unambiguously determine its further trajectory without additional information. In combination with the EM reconstruction data, however, sometimes further conclusions
can be drawn. An example, where a large number of
Fig. 3. Axon trajectories into the nerve ring. Interneuron cell
bodies and axons labelled with GFP under the control of the glr-1
promoter. Pictures in multiple focal planes were recorded with a
confocal microscope and deconvolved with Huygens2. Arrowheads
point to the axons in question. A–D: Head region of an animal showing
the AVJL axon projecting into the nerve ring. Side view, anterior is to
the left. The AVJL axon first projects anteriorly from the cell body,
enters the nerve ring subdorsally and immediately turns dorsally
(arrowheads). A: Maximum intensity projection of 14 focal planes
taken at 120-nm intervals illustrating the entire axon trajectory. Scale
bar ⫽ 5 µm (valid for all panels except panel G). B–D: Single focal
plane images. E: Dorsal view of a different animal illustrating the
AVJL and AVJR axon trajectories from the dorsal side. Despite the
large number of labelled axons in the ring the AVJ axons can be clearly
followed as they enter the ring and stay near the posterior end of the
ring while growing dorsally (arrowheads). F: The RIML neuron, whose
cell body is located close to the AVJL cell body, sends its axon into the
nerve ring by a rather different route. It first grows ventrally towards
the ventral cord and enters the nerve ring from the ventral side. G:
The RMDL cell body is located close the nerve ring. Its axon also
extends into the ring but in contrast to the AVJL axon grows through
the ring past the axons that the AVJL axon joins after turning dorsally
(small arrow) Scale bar ⫽ 2 µm. H: Closeup of the dorsalmost part of
the nerve ring, dorsal view, anterior to the left. One pair of axons
growing dorsally near the posterior end of the nerve ring abruptly
change directions at the dorsal midline and continue to grow on the
contralateral side near the anterior end of the ring. These axons most
probably are the AVEL and AVER axons, since those are the only ones
among the GFP-labelled ones that are known for this kind of behaviour from EM reconstructions.
axons in the nerve ring are GFP-labelled, is shown in
Figure 3. At the dorsal midline, a pair of axons abruptly
changes its direction of growth (Fig. 3H). It is impossible to trace these axons back to their cell bodies since
other labelled axons run close to them in the nerve ring.
However, from the EM reconstructions we know that
among the labelled axons only the two AVE axons
(AVEL and AVER) exhibit such a prominent change of
direction at the dorsal midline. These axons, therefore,
are likely to be the AVER and AVEL axons. The
observations made with the GFP reporter can serve as a
valuable basis for a more detailed analysis. Doublelabelling experiments would be necessary to validate
and extend the observations made with the single label.
However, EM reconstructions still are the method of
choice for a complete reconstruction of many different
axon trajectories within a single animal in C. elegans.
Although it is possible to separate GFP and CFP
under optimal conditions, this is not the ideal combination for double labeling where clean separation of the
signals is required. A combination of the cyan variant of
GFP together with a yellow shifted variant (10Cvariant; Ormö et al., 1996) allows an easier separation with
a simple microscope setup (see Ellenberg et al., 1998,
1999 for a summary of GFP variants used in double
color imaging). This combination has recently been
used successfully to label different subcellular structures in C. elegans (Miller et al., 1999). The overlapping
exciation and emission spectra of CFP and GFP, which
makes it difficult to separate them, are advantageous
when two colors are needed in rapid time lapse recording experiments, since CFP and GFP can be excited and
recorded simultaneously on a confocal microscope (Zimmermann and Siegert, 1998).
GFP markers are also used successfully in other
organisms to improve the resolution in axonal imaging
and as a label for particular neurons in living tissue
(Rodriguez et al., 1999). The spacial and temporal
resolution that is achived with GFP enables researchers now to study in vivo the changes in the dendritic
architecture in response to individual factors (Horch et
al., 1999) or even the dynamics at a single synapse as it
grows (Zito et al., 1999). These and many other studies
will soon lead to a deeper understanding of developmental processes in the brain.
Towards the Isolation of Genes Important for the
Generation of Neuronal Circuits
In their initial screens, Brenner and co-workers
(Brenner, 1974) isolated a large number of mutants
that were characterised by specific behavioural defects.
The most prominent behavioural defects they scored
were movement defects. The corresponding genes were
called unc-genes, a reference to the ‘‘uncoordinated’’
movement of the affected animals. Since then a number
of researchers have collected many mutants in genes
affecting all aspects of behaviour in C. elegans with the
aim of understanding various aspects of neuronal development.
The collection of unc-genes now consists of 130 genes,
many of which have been studied in detail. Among
those, about one-third is important for various aspects
of muscle development (Moerman and Fire, 1997),
whereas the majority affects neuronal development and
function (Riddle et al., 1997). Among the neurological
mutants many play a role in synaptic transmission and
encode ion-channels, enzymes involved in neurotransmitter metabolism, or proteins important for neurotransmitter release. A smaller number of neurological
mutants have defects in various aspects of neuronal
development most notably neuronal cell fate specification and axonal guidance. The fraction of unc-genes
involved in this latter aspect is less than 20%, which
illustrates that a purely behavioural screen is not the
most effective way to identify genes important for
axonal guidance. Evaluating the movement of an animal is a rather indirect way of judging the developmental state of its nervous system. With GFP-markers more
direct screens for neurological mutants, even such with
only subtle defects in axonal outgrowth are now possible.
The ability to perform double-labelling experiments
in the living animal and the high spatial resolution
achieved with modern microscopy techniques will allow
us to isolate and analyse genes with important roles in
aspects of neuronal development that were not easily
accessible so far. In particular, local guidance signals
that are used only by a subset of neurons and signals
that are used for synaptic target recognition can now be
identified through genetic screens. Our laboratory has
started to screen for mutants with defects in the
fasciculation of interneuron axons in the ventral cord.
We have collected a first set of mutants with defects in
the navigation of these axons within the ventral cord
(Hutter and Hedgecock, unpublished data). Some of
these mutants show outgrowth defects that do not lead
to pronounced behavioural defects and would not have
been found in traditional behavioural screens or in
screens that rely on histochemical markers.
I thank J. Kaplan, D. Baillie, and A. Fire for vectors,
J. Culotti for an unc-129::GFP strain, S. Liebe and U.
Tauer at LEICA Microsystems Heidelberg GmbH for
their help with taking images at their LEICA SP
confocal microscope, G. Giese for an introduction to the
Huygens system, and I. Wacker for critical reading of
the manuscript.
Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC. 1994. Green
fluorescent protein as a marker for gene expression. Science 263:802–
Ellenberg J, Lippincott-Schwartz J, Presley JF. 1998. Two-color green
fluorescent protein time-lapse imaging. Biotechniques 25:838–846.
Ellenberg J, Lippincott-Schwartz J, Presley JF. 1999. Dual-colour
imaging with GFP variants. Trends Cell Biol 9:52–56.
Hart AC, Sims S, Kaplan J. 1995. Synaptic code for sensory modalities
revealed by C. elegans glr–1 glutamate receptor. Nature 378:82–85.
Hedgecock EM, Culotti JG, Hall DH. 1990. The unc–5, unc–6, and
unc–40 genes guide circumferential migrations of pioneer axons and
mesodermal cells on the epidermis in C. elegans. Neuron 2:61–85.
Hedgecock EM, Culotti JG, Thomson JN, Perkins LA. 1985. Axonal
guidance mutants of Caenorhabditis elegans identified by filling
sensory neurons with fluorescein dyes. Dev Biol 111:158–170.
Heim R, Tsien RY. 1996. Engineering green fluorescent protein for
improved brightness, longer wavelength and fluorescence resonance
energy transfer. Curr Biol 6:178–182.
Horch HW, Kruttgen A, Portbury SD, Katz LC. 1999. Destabilization
of cortical dendrites and spines by BDNF. Neuron 23:353–364.
Ishii N, Wadsworth WG, Stern BD, Culotti JG, Hedgecock E M. 1992.
UNC–6, a laminin-related protein, guides cell and pioneer axon
migrations in C. elegans. Neuron 9:873–881.
McIntire SL, Garriga G, White J, Jacobson D, Horvitz HR. 1992.
Genes necessary for directed axonal elongation or fasciculation in C.
elegans. Neuron 8:307–322.
McIntire SL, Jorgensen E, Kaplan J, Horvitz HR. 1993. The
GABAergic nervous system of Caenorhabditis elegans. Nature 364:
Mello C, Fire A. 1995. DNA transformation. In: Epstein HF, Shakes
DC, editors. Caenorhabditis elegans: modern biological analysis of
an organism. San Diego: Academic Press. 48:451–482.
Miller DM, Desai NS, Hardin DC, Piston DW, Patterson GH, Fleenor
J, Xu S, Fire A. 1999. Two-color GFP expression system for C.
elegans. BioTechniques 26:914–921.
Moerman DG, Fire A. 1997. Muscle: structure function, and development. In: Riddle DL, Blumenthal T, Meyer BJ, Priess JR, editors. C.
elegans II. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press. p 417–470.
Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ.
1996. Crystal structure of the Aequorea victoria green fluorescent
protein. Science 273:1392–1395.
Riddle DL, Blumenthal T, Meyer BJ, Priess JR. 1997. C. elegans II.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Rodriguez I, Feinstein P, Mombaerts P. 1999. Variable patterns of
axonal projections of sensory neurons in the mouse vomeronasal
system. Cell 97:199–208.
Siddiqui SS, Culotti JG. 1991. Examination of neurons in wild type
and mutants of caenorhabditis elegans using antibodies to horseradish peroxidase. J Neurogenet 7:193–211.
Snyder DL, Schulz TJ, O’Sullivan JA. 1992. Deblurring subject to
nonnegativity constraints. IEEE Trans Sign Proc 40:1143–1150.
Sulston JE, Horvitz HR. 1977. Post-embryonic cell lineages of the
nematode Caenorhabditis elegans. Dev Biol 56:110–156.
Sulston JE, Schierenberg E, White JG, Thomson JN. 1983. The
embryonic cell lineage of the nematode Caenorhabditis elegans. Dev
Biol 100:64–119.
Wadsworth WG, Bhatt H, Hedgecock EM. 1996. Neuroglia and pioneer
neurons express UNC–6 to provide global and local netrin cues for
guiding migrations in C. elegans. Neuron 16:35–46.
White J, Southgate E, Thomson J, Brenner S. 1986. The structure of
the nervous system of the nematode Caenorhabditis elegans. Phil
Trans R Soc Lond B. 314:1–340.
White JG, Southgate E, Thomson JN, Brenner S. 1976. The structure
of the ventral nerve cord of Caenorhabditis elegans. Phil Trans R Soc
Lond B 275:327–348.
Zimmermann T, Siegert F. 1998. Simultaneous detection of two GFP
spectral mutants during in vivo confocal microscopy of migrating
Dictyostelium cells. Biotechniques 24:458–461.
Zito K, Parnas D, Fetter RD, Isacoff EY, Goodman CS. 1999. Watching
a synapse grow: noninvasive confocal imaging of synaptic growth in
Drosophila. Neuron 22:719–29.
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