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Wiring the zebrafish: axon guidance and synaptogenesis
Lara D Hutson and Chi-Bin Chien*
Many zebrafish mutants have specific defects in axon guidance
or synaptogenesis, particularly in the retinotectal and motor
systems. Several mutants have now been characterized in
detail and/or cloned. A combination of genetic studies, in vivo
imaging and new techniques for misexpressing genes or
blocking their function promises to reveal the molecules and
principles that govern wiring of the vertebrate nervous system.
Addresses
Department of Neurobiology and Anatomy, Room 401 Medical
Research and Engineering Building, 20 North 1900 East,
University of Utah Medical Center, Salt Lake City, Utah 84132, USA
*e-mail: [email protected]
Current Opinion in Neurobiology 2002, 12:87–92
0959-4388/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
Abbreviations
ace
acerebellar
ast
astray
CaP
caudal primary motor neuron
diw
diwanka
GFP
green fluorescent protein
HMS
horizontal myoseptum
mao
macho
MiP
middle primary motor neuron
noi
no isthmus
RGC
retinal ganglion cell
Robo
Roundabout
RoP
rostral primary motor neuron
Sema semaphorin
smu
smooth-muscle-omitted
sop
sofa potato
spc
space cadet
sty
stumpy
syu
sonic-you/sonic hedgehog
unp
unplugged
yot
you-too/gli2
Introduction
The past decade has brought great strides in our molecular
understanding of axon guidance and synaptogenesis, particularly through genetic analysis of invertebrate models and
in vitro analysis of vertebrates. Such analyses have revealed
an apparently canonical set of gene families that are conserved across the animal kingdom [1]. Given this knowledge,
three questions stand out. First, how do the known genes act
in the animal to guide particular axons? Second, how many
axon guidance genes remain to be found? Third, does the
relative complexity of the vertebrate nervous system arise
from using old gene families in new ways, or by virtue of vertebrate-specific gene families? Studying the zebrafish
promises to yield answers to these questions.
The zebrafish embryo has several well-known experimental
advantages. For example, external fertilization provides early
embryonic access. Its nervous system is simple and
well-characterized (reviewed in [2,3•]). Its optical transparency
allows in vivo imaging of cell movements (e.g. see [4]) or
ablation of identified cells to perturb development or behavior
[5,6]. Finally, cells or tissues can be transplanted to test the
autonomy of gene action (e.g. see [7,8••]).
Recent genetic screens have identified many genes necessary
for axon pathfinding and synaptogenesis [9–12]. It has now
become relatively routine to clone mutant genes, by using
candidate gene, positional cloning or insertional mutagenesis
approaches [13]. Three new technical advances show particular promise for analyzing gene function: the development of
transgenic lines that express green fluorescent protein (GFP)
in specific sets of neurons [14•]; the use of a heat-shock
promoter to misexpress genes in specific cells [15•]; and the
use of antisense morpholino oligonucleotides — whose
modified backbone provides increased stability and reduced
toxicity — to knock out gene function. [16•].
In this review, we discuss recent studies that use this
sizeable bag of tricks to analyze how axons find and form
synapses with their targets, particularly in the retinotectal
and motor systems.
Retinotectal pathfinding
Most retinal ganglion cell (RGC) axons project to the
primary visual center in fish — the contralateral optic tectum
[17,18] (Figure 1a). Retinal axons project to the optic nerve
head in central retina and exit the eye. They then grow
along the ventral surface of the optic stalk (which later
becomes the optic nerve), cross the ventral midline of the
forebrain, turn dorsocaudally, and project to the optic
tectum in the dorsal midbrain. There, they branch extensively, terminating topographically according to their point
of origin in the retina.
In several species, specific guidance molecules are known
to function in the eye (e.g. netrin/DCC [deleted in colorectal
cancer]), at the midline (GAP-43 [growth-associated
protein-43], ephrinB/EphBs), and along the anteroposterior
axis of the tectum (ephrinA/EphAs) (reviewed in [19–21]),
but many segments of the pathway remain unexplained
molecularly. A direct screen for retinotectal projection
defects has yielded a large set of mutants, each with specific
defects at one or two points [10,22,23] (Figure 1b–h).
Many of these perturb brain or eye patterning, whereas
others may affect axon guidance more directly.
Mutants with mispatterned brains
In midline-crossing mutants (Figure 1d), retinal axons
project to both the ipsilateral and contralateral optic tecta,
forebrain commissures are often defective, and the forebrain
is often narrowed [10,24–27]. The three cloned mutants in
this class, sonic-you/sonic hedgehog (syu) [28], you-too/gli2 (yot)
[25] and smooth-muscle-omitted (smu)/smoothened (smo) [26,27],
88
Development
Figure 1
(a) Wild type
(b) Retinal exit
(c) Elongation
(d) Midline crossing
A
A
V
D
bal, con, spc, syu
P
esr, spc
P
(e) Pathfinding to tectum
ace, bal, gup,
noi, sly
(f) Tract sorting
ast
box, dak, pic
(g) Topography
ace, nev, woe
bel, blw, con, dtr, igu,
smu, spc, syu, uml, yot
(h) Termination
mao, blu, gna
Current Opinion in Neurobiology
Mutations disrupting different steps in formation of the retinotectal
projection. (a) Dorsal view (right) of wild-type projection at 5 days
postfertilization, after labeling groups of RGCs in the dorsoanterior
(green) and ventroposterior (red) quadrants of the eye. Labeling is
shown in the corresponding lateral view (left). Retinal axons project out
of the eye, across the midline and to the contralateral optic tectum.
They are topographically ordered as they exit the eye, rearrange this
order shortly after the midline and terminate topographically on the
tectum. (b–h) Dorsal views of mutant phenotypes. (b) In four mutants,
bashful (bal), chameleon (con), spc and syu, axons sometimes fail to
exit the eye. (c) Two mutants, esrom (esr) and spc have defects in
elongation, with few axons reaching the tectum. (d) Ten mutants,
belladonna (bel), blowout (blw), con, detour (dtr), iguana (igu), smu,
spc, syu, umleitung (uml) and yot have defects in midline crossing,
with projections to both ipsilateral and contralateral tecta. (e) Six
mutants, ace, bal, grumpy (gup) noi, sleepy (sly) and ast, have defects
in pathfinding to the tectum, with ipsilateral and anterior projections. In
noi, retinal axons also project into the opposite eye. In ast, retinal axons
also project into the opposite eye and to ventral hindbrain, and show
extra midline crossing. (f) Three mutants, boxer (box), dackel (dak) and
pinscher (pic), have defects in optic tract sorting, but normal tectal
topography; box and pic also have projections to ipsilateral tectum.
(g) Three mutants, ace, nevermind (nev) and who-cares (woe), have
defects in optic tract sorting and defective tectal topography: along
the dorsoventral axis for nev and woe, and along both axes for ace.
(h) Three mutants, mao, blumenkohl (blu) and gnarled (gna), show
expanded or defective tectal terminations. Orange font indicates that
the mutant gene has been cloned; underlined names indicate a
known defect in brain patterning. A: anterior; D: dorsal; P: posterior;
V: ventral.
all function in the Hedgehog pathway, which patterns the
midline. The smu mutant shows decreased expression of
netrin1 in the forebrain [27], which might affect the axon tracts.
In general, the retinotectal pathfinding errors in these mutants
are probably secondary to defects in forebrain patterning.
Retinal axons gone astray
In another class of mutants, retinal axons project anteriorly in
the forebrain and to ipsilateral tectum (Figure 1e). Two of
these mutants, no isthmus/pax2.1 (noi) and acerebellar/fgf8 (ace)
mutants have been shown to affect not only early midbrain
patterning (as their names imply) but also forebrain patterning [29,30•]. As both have defective forebrain commissures,
might ace and noi regulate forebrain guidance signals that are
used in common by retinal and forebrain commissural axons?
In ace mutants, netrin1 and sema3D (formerly semaZ2) are
downregulated, whereas in noi, ephrinA5a and ephrinA5b
(formerly L4 and L2) mutants, netrin1 and netrin2 are
upregulated [29,30•]. It is not known, however, whether
these changes cause the observed retinal projection defects.
The astray/robo2 (ast) mutant has the most dramatic known
retinotectal phenotype. In addition to mistakes similar to
ace and noi, ast retinal axons project into ventral hindbrain
and recross both the ventral and dorsal midlines [8••,10].
Brain patterning and the forebrain commissures appear
normal, however, and eye transplants show that ast acts
eye-autonomously. Cloning of ast shows that it is a defect
in Robo2 — a homolog of the Drosophila axon guidance
receptor Roundabout (Robo) [8••,31] — which is expressed
in RGCs during axon outgrowth. As robo2 is also expressed
in mouse RGCs [32–34], it is likely to be essential for
retinal axon guidance in all vertebrates.
We have used high-resolution in vivo imaging of growth cone
behavior to try and elucidate the function of ast. We found
that ast growth cones make frequent pathfinding errors and
fail to correct them, whereas wild-type growth cones make
occasional pathfinding errors (to our surprise), but always
Wiring the zebrafish: axon guidance and synaptogenesis Hutson and Chien
correct them. Importantly, ast affects retinal growth cone
morphology, behavior and pathfinding before, at and after
the midline (LD Hutson, C-B Chien, unpublished data).
This contrasts with Drosophila commissural axons. In these
axons, Robo is downregulated (and therefore nonfunctional)
at the midline, and pathfinding defects in robo mutants are
only seen after midline crossing [31]. What accounts for this
difference? Perhaps it is because Robo ligands are
expressed in different patterns. In Drosophila, commissural
axons must cross a midline band of Slit, a repulsive Robo
ligand [35]; in contrast, vertebrate slits are expressed in
domains adjacent to, but not crossing, the retinal axon pathway [32–34] (Hutson LD, C-B Chien, unpublished data).
Retinotectal plus: space cadet and macho
Originally, space cadet (spc) mutants were described as having
two disparate phenotypes: retinotectal pathfinding defects
and faulty escape behavior [9,36••] (Figures 1b–d,2). Recent
work has shown that these defects may share a common
basis. Normally, spiral fiber neurons form synapses on
Mauthner neurons (large hindbrain neurons that control
the escape response), presumably modulating their activity
(Figure 2a). In spc mutants, the spiral fibers do not innervate
the Mauthner neurons (Figure 2b), and the spc behavioral
defect can be phenocopied by microsurgical transection of
axon tracts that include the spiral fibers (Figure 2a).
Therefore, spc may be required for axon pathfinding of
spiral fibers as well as retinal axons.
The macho (mao) mutant also has apparently disparate
phenotypes: enlarged retinotectal arbors and touch insensitivity [9,23]; (Figure 1h). These have been shown to have a
common biophysical underpinning. The touch-response
defect is caused by lack of sodium current in the sensory
Rohon–Beard cells [37]. mao RGCs also lack sodium
current, and injecting the sodium-channel blocker tetrodotoxin into the eye or the optic tectum can phenocopy the
enlargement of arbors seen in mao mutants [38••]. Thus, mao
provides a genetic recapitulation of classic tetrodotoxinblocking experiments [39], and may prove useful for studying
other activity-dependent processes.
Motor axon pathfinding
Zebrafish trunk motor neurons develop in two waves. In
each hemisegment, three identified primary motor neurons,
named CaP, MiP and RoP (for caudal, middle and rostral
primary), are formed 9–10 hours after fertilization, and
about 30 secondary motor neurons are formed a few hours
later [3•]. All three primary motor neurons extend their
axons within the spinal cord to a shared exit point, and
then follow a shared ‘common pathway’ to reach the
horizontal myoseptum (HMS), which is marked by a set of
specialized muscle pioneer cells. Here they pause before
diverging to distinct targets (Figure 3a,b).
The cellular interactions used for CaP/MiP/RoP axon
guidance have been studied extensively [3•]. Each
89
Figure 2
(a) Wild type
R
r3
r4
C
1
(b) Space cadet
2
s
s
s
s
M
ov
?
M
ov
r5
Current Opinion in Neurobiology
The development of spiral fiber axons is disrupted in spc mutants.
Dorsal views of hindbrain in wild type (a) and spc (b) embryos. Rostral
is up. (a) In wild-type, spiral fiber axons (s) cross the midline in
rhombomere 3 (r3) and project caudally to synapse on the initial
segments of the Mauthner axons (M) in rhombomere 4 (r4).
Transections (indicated in green) of either the r3 commissures (1) or
the longitudinal tracts between r3 and r4 (2) can phenocopy the spc
behavioral defect. (b) In spc mutants, spiral fiber axons fail to make
their appropriate projections onto the Mauthner neurons. It is not
known whether they project inappropriately, or perhaps fail to develop
altogether. ov: otic vesicle; C: caudal; R: rostral.
primary motor neuron can project correctly even with the
other two ablated, but their axons do require myotomederived signals. Although the secondary motor axons can
navigate correctly when the primary motor neurons are
ablated [5], they do follow the paths of the primary
axons [4], and perturbations that affect the primary
axons can also affect the secondary axons [40 •].
Therefore, primary and secondary motor axons may
share some pathfinding mechanisms.
Several axon guidance molecules are known to be
expressed by the motor neurons and myotome [3•]. Among
these, chondroitin sulfate proteoglycans [41], GDNF (glial
cell line derived neurotrophic factor) [42], semaphorin
(Sema) 3A1 (formerly SemaZ1a) [15•] and Sema3A2
(formerly SemaZ1b) [43] can impede ventral motor axon
outgrowth when injected or overexpressed. In addition,
enzymatic removal of chondroitin sulfate proteoglycans
[41] and function-blocking antibodies to Neurolin [44]
cause aberrant branching and outgrowth of primary or
secondary motor axons, respectively. More definitive
experiments are required before the roles of these molecules
in motor axon guidance are understood.
Motor axon mutants
Three known mutants specifically disrupt primary motor
axon pathfinding without affecting muscle, notochord or
floor plate development [9,12]. diwanka (diw) and
unplugged (unp) were found using antibody staining to
re-screen mutants found in a large-scale embryonic motility
screen, whereas stumpy (sty) was found in a smaller screen
that used antibody staining as the primary assay. Each
mutant has a distinct defect in motor axon navigation
(Figure 3c–e), suggesting that they disrupt different
signaling pathways.
90
Development
Figure 3
Mutations that specifically disrupt pathfinding
by primary motor axons. (a,b) (a) Transverse
and (b) lateral views showing wild-type
projections of the three primary motor neurons,
RoP (R; green), MiP (M; red) and CaP (C; blue),
in each hemisegment. Axons from RoP, MiP
and CaP project ventrally from the spinal cord
(sc) along the common pathway to the HMS,
and then diverge. RoP invades the myotome at
the level of the HMS, whereas MiP retracts its
common pathway projection and instead
projects to dorsal myotome. CaP continues on
and projects to ventral myotome. Not shown is
VaP, the variable primary motor neuron, which
is present in about 50% of segments and
usually dies. Also shown in (a) are the adaxial
cells (orange), which have an essential role in
guiding motor axons. These cells originate
adjacent to the notochord (not), migrate
laterally and eventually differentiate to form
slow muscle fibers. (c–e) Lateral views of
(a) Wild type
D
sc
sc
M
R
C
(c) diwanka
(d) unplugged
M
R
M
R
C
(e) stumpy
M
R
C
C
not
not
V
(b) Wild type
som
som
HMS
Rostral
Caudal
Current Opinion in Neurobiology
mutant phenotypes. (c) In diw, all three
primary motor axons either fail to project into,
or stall within, the common pathway. RoP
occasionally bypasses the spinal cord exit
point altogether, projecting caudally within the
spinal cord. (d) In unp, CaP often stalls and
extends aberrant branches at the HMS.
Occasionally, RoP also branches aberrantly at
The diw mutants show accordion-like contraction in
response to a touch stimulus, rather than the side-to-side tail
motion seen in wild-type [9], and all three primary motor
axons show defects (Figure 3c). Most RoPs arrest at the
spinal cord exit point and fail to enter the common pathway,
whereas most MiPs and CaPs stall along the common
pathway before reaching the HMS [7]. Analyzing genetic
mosaics created by cell transplantation shows that diw function is required in the adaxial cells — a set of slow-muscle
precursors whose path of migration seems to prefigure the
common pathway [7]. This suggests that adaxial cells either
secrete, or instruct the somite to produce, a signal that
attracts growth cones along the common path.
The unp mutants are initially immotile but can later swim
[9]. All three primary motor axons exit the spinal cord and
pathfind correctly to the HMS, but thereafter CaP and
RoP fail to follow their normal pathways, whereas MiP
projects normally. Most CaP axons, which normally project
to ventral myotome, stall at the HMS, occasionally forming
inappropriate branches. Most RoP axons, which normally
pause at the HMS, either branch abnormally or project
into the ventral myotome. Secondary motor axons show
defects very similar to those of CaP and RoP [40•]
(Figure 3d). Transplant experiments show that like diw,
unp is required in adaxial cells [45••], suggesting that
adaxial cells help to specify not only the common but also
the cell type-specific pathways.
In sty mutants, the common pathway appears normal, but
extension of the primary motor neurons along their
individual pathways, together with secondary motor axon
extension, is affected [12]. In spite of these defects, sty has
no obvious motility defect. In a strong sty allele, most CaP
axons stall at the HMS, whereas the rest stall before
reaching their targets in ventral myotome. MiP and RoP
the HMS, sometimes sending one projection
into ventral myotome. MiP projects normally.
(e) In sty, CaP initially stalls at the HMS, but
then sometimes extends further into the ventral
myotome. MiP and RoP initially project
normally, but later show subtle defects such as
reduced branching. som: somite; D: dorsal;
V: ventral.
axons in sty pathfind correctly to the HMS but
subsequently exhibit various branching and pathfinding
defects [12]; (Figure 3e). Transplant experiments show
that sty is required both in the motor neurons and in the
surrounding cells, suggesting that Stumpy might be a
homophilic cell-surface protein [46••].
Visualizing synaptogenesis
Two recent studies have analyzed synaptogenesis in vivo.
Jontes et al. [47] used confocal time-lapse microscopy to
visualize encounters between Mauthner axons and their
motor neuron targets. They showed that the presynaptic
axon does not always play an active role; instead, synapses
sometimes form after dendritic filopodia from the postsynaptic motor neuron reach out to contact a passing
Mauthner axon.
Ono et al. [48•] have looked at motility mutants from a
class that completely lacks muscular contraction [9]; their
results suggest that these mutants have defects in muscle
function or neuromuscular transmission. Muscles in sofa
potato (sop) lack functional nicotinic acetylcholine receptors [48•]. The in vivo function of rapsyn, which clusters
acetylcholine receptors, has been tested using a transgenic
line that expresses a rapsyn–GFP fusion in muscles. In sop
mutants, rapsyn–GFP forms clusters but does not localize
to synapses, suggesting that acetylcholine receptors themselves are required to localize rapsyn to the synapse [48•].
Such in vivo imaging will be useful indeed for further
studies of synaptogenesis.
Conclusions
The zebrafish retinotectal and primary motor axon projections have revealed much about the tissue interactions and
cellular behavior that underlie their axon pathfinding.
Studies of retinotectal and motor synaptogenesis have just
Wiring the zebrafish: axon guidance and synaptogenesis Hutson and Chien
begun, but are promising. Other systems such as spinal
cord interneurons and the lateral line [49,50] will also be
useful, especially once particular neurons can be lit up
with GFP. To this end, many groups are isolating cell-typespecific promoters. As in that other transparent organism,
Caenorhabditis elegans, GFP transgenics will be powerful
tools for both analyzing old mutants and finding new ones.
The molecular mechanisms underlying zebrafish pathfinding
and synaptogenesis are starting to be understood, especially
as the mutants are cloned. Vigorous efforts to clone the
remaining pathfinding mutants are underway in several
laboratories, and will be helped greatly by the sequence
of the zebrafish genome, which is scheduled to be fully
completed by late 2003.
5.
Eisen JS, Pike SH, Debu B: The growth cones of identified
motoneurons in embryonic zebrafish select appropriate pathways
in the absence of specific cellular interactions. Neuron 1989,
2:1097-1104.
6.
Liu KS, Fetcho JR: Laser ablations reveal functional relationships
of segmental hindbrain neurons in zebrafish. Neuron 1999,
23:325-335.
7.
Zeller J, Granato M: The zebrafish diwanka gene controls an early
step of motor growth cone migration. Development 1999,
126:3461-3472.
8.
••
Fricke C, Lee JS, Geiger-Rudolph S, Bonhoeffer F, Chien CB: Astray,
a zebrafish Roundabout homolog required for retinal axon
guidance. Science 2001, 292:507-510.
The retinotectal pathfinding mutant astray is characterized in detail and
cloned. Eye transplants show that astray function is required in the eye. The
gene is the axon guidance receptor robo2, a homolog of fruitfly roundabout,
and its mRNA is expressed transiently in RGCs during the period of retinal
axon pathfinding. astray is the first known mutation in a zebrafish axon guidance receptor or ligand, as well as the only vertebrate Robo mutant to date.
9.
Granato M, van Eeden FJ, Schach U, Trowe T, Brand M, FurutaniSeiki M, Haffter P, Hammerschmidt M, Heisenberg CP, Jiang YJ et al.:
Genes controlling and mediating locomotion behavior of the
zebrafish embryo and larva. Development 1996, 123:399-413.
We predict that nearly all the remaining pathfinding
mutants will be cloned within the next two years. As
mutant cloning becomes less of a hurdle, zebrafish
researchers can concentrate on analyzing existing mutants
and screening for new ones. The genetic, molecular and
imaging tools available in zebrafish will allow us to discover
how our favorite genes really work in vivo, and how they
contribute to the wiring of the vertebrate nervous system.
11. Neuhauss SC, Biehlmaier O, Seeliger MW, Das T, Kohler K,
Harris WA, Baier H: Genetic disorders of vision revealed by a
behavioral screen of 400 essential loci in zebrafish. J Neurosci
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Update
12. Beattie CE, Raible DW, Henion PD, Eisen JS: Early pressure
screens. Methods Cell Biol 1999, 60:71-86.
The data demonstrating that ast is required for both
preventing and correcting small-scale pathfinding errors by
retinal axons throughout the ventral forebrain are now in
press [51••]. Also described in this paper are the forebrain
expression patterns of the putative Robo2 ligands, slit2 and
slit3. Both slits are expressed on one or both sides of the
retinotectal pathway, consistent with a role in shaping the
pathway through the forebrain, presumably by signaling
through ast.
Acknowledgements
Thanks to our zebrafish colleagues for illuminating discussions and for
sharing unpublished data; we apologize to those whose work has been
omitted owing to space limitations. The authors are supported by the
National Institutes of Health (grant F32-EY07017 to LD Hutson, and
R01-EY12873 to C-B Chien).
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Varga ZM, Amores A, Lewis KE, Yan YL, Postlethwait JH, Eisen JS,
Westerfield M: Zebrafish smoothened functions in ventral neural
tube specification and axon tract formation. Development 2001,
128:3497-3509.
28. Schauerte HE, van Eeden FJ, Fricke C, Odenthal J, Strahle U,
Haffter P: Sonic hedgehog is not required for the induction of
medial floor plate cells in the zebrafish. Development 1998,
125:2983-2993.
29. Macdonald R, Scholes J, Strahle U, Brennan C, Holder N, Brand M,
Wilson SW: The Pax protein Noi is required for commissural axon
pathway formation in the rostral forebrain. Development 1997,
124:2397-2408.
30. Shanmugalingam S, Houart C, Picker A, Reifers F, Macdonald R,
•
Barth A, Griffin K, Brand M, Wilson SW: Ace/Fgf8 is required for
forebrain commissure formation and patterning of the
telencephalon. Development 2000, 127:2549-2561.
The authors of this paper analyze the forebrain phenotype of ace. ace
mutants have axon pathfinding defects, as well as altered expression of several axon guidance signals and patterning genes (including noi/pax2.1).
31. Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M,
Goodman CS, Tear G: Roundabout controls axon crossing of the
CNS midline and defines a novel subfamily of evolutionarily
conserved guidance receptors. Cell 1998, 92:205-215.
32. Erskine L, Williams SE, Brose K, Kidd T, Rachel RA, Goodman CS,
Tessier-Lavigne M, Mason CA: Retinal ganglion cell axon guidance
in the mouse optic chiasm: expression and function of Robos and
Slits. J Neurosci 2000, 20:4975-4982.
33. Niclou SP, Jia L, Raper JA: Slit2 is a repellent for retinal ganglion
cell axons. J Neurosci 2000, 20:4962-4974.
34. Ringstedt T, Braisted JE, Brose K, Kidd T, Goodman C, TessierLavigne M, O’Leary DD: Slit inhibition of retinal axon growth and its
role in retinal axon pathfinding and innervation patterns in the
diencephalon. J Neurosci 2000, 20:4983-4991.
35. Kidd T, Bland KS, Goodman CS: Slit is the midline repellent for the
Robo receptor in Drosophila. Cell 1999, 96:785-794.
36. Lorent K, Liu KS, Fetcho JR, Granato M: The zebrafish space cadet
•• gene controls axonal pathfinding of neurons that modulate fast
turning movements. Development 2001, 128:2131-2142.
Laurent et al. provide a nice example of the diverse techniques that can be
applied to zebrafish, including high-speed behavioral analysis, confocal
microscopy and embryological manipulations. Using a clever trans-synaptic
labeling technique, the authors show that the spiral fibers form synapses
with Mauthner neurons and that their projections are disrupted in the spc
mutant. Transecting these axons phenocopies spc, suggesting that their
absence causes the defect in the spc escape response.
37.
Ribera AB, Nusslein-Volhard C: Zebrafish touch-insensitive
mutants reveal an essential role for the developmental regulation
of sodium current. J Neurosci 1998, 18:9181-9191.
38. Gnuegge L, Schmid S, Neuhauss SC: Analysis of the
•• activity-deprived zebrafish mutant macho reveals an essential
requirement of neuronal activity for the development of a finegrained visuotopic map. J Neurosci 2001, 21:3542-3548.
The authors show that enlarged retinotectal arbors in mao mutants are due
to an activity-dependent defect. Perforated-patch recording shows that
RGCs lack almost all sodium current, and tetrodotoxin injections can phenocopy the enlarged arbor phenotype.
39. Penn AA, Shatz CJ: Brain waves and brain wiring: the role of
endogenous and sensory-driven neural activity in development.
Pediatr Res 1999, 45:447-458.
40. Zhang J, Malayaman S, Davis C, Granato M: A dual role for the
•
zebrafish unplugged gene in motor axon pathfinding and
pharyngeal development. Dev Biol 2001, 240:560-573.
A further analysis of unp (see [45••]) shows that there is also a secondary
motor phenotype that is quite similar to the CaP/RoP phenotype. Intriguingly,
there is also a subtle jaw phenotype, suggesting that unp may act in cell
migration as well as axon guidance.
41. Bernhardt RR, Schachner M: Chondroitin sulfates affect the
formation of the segmental motor nerves in zebrafish embryos.
Dev Biol 2000, 221:206-219.
42. Shepherd IT, Beattie CE, Raible DW: Functional analysis of
zebrafish GDNF. Dev Biol 2001, 231:420-435.
43. Roos M, Schachner M, Bernhardt RR: Zebrafish semaphorin Z1b
inhibits growing motor axons in vivo. Mech Dev 1999, 87:103-117.
44. Ott H, Diekmann H, Stuermer CA, Bastmeyer M: Function of neurolin
(dm-grasp/sc-1) in guidance of motor axons during zebrafish
development. Dev Biol 2001, 235:86-97.
45. Zhang J, Granato M: The zebrafish unplugged gene controls motor
•• axon pathway selection. Development 2000, 127:2099-2111.
A detailed analysis of unplugged shows that it is specifically required for
CaP and RoP axon pathfinding along their cell-type-specific pathways. As for
diw (see [7]), transplant experiments show that unp function is required in
the adaxial cells, emphasizing their importance for motor axon guidance.
46. Beattie CE, Melancon E, Eisen JS: Mutations in the stumpy gene
•• reveal intermediate targets for zebrafish motor axons.
Development 2000, 127:2653-2662.
Here, the sty mutant is analyzed in detail. It shows specific defects in primary and secondary motor axons, especially CaP axons. These axons stall at
two specific points, which seem to be intermediate targets where the axons
need sty function to grow further. Transplants show that sty is required both
in axons and in neighboring cells.
47.
Jontes JD, Buchanan J, Smith SJ: Growth cone and dendrite
dynamics in zebrafish embryos: early events in synaptogenesis
imaged in vivo. Nat Neurosci 2000, 3:231-237.
48. Ono F, Higashijima S, Shcherbatko A, Fetcho JR, Brehm P: Paralytic
•
zebrafish lacking acetylcholine receptors fail to localize rapsyn
clusters to the synapse. J Neurosci 2001, 21:5439-5448.
The authors start to analyze the paralyzed mutants, a class that has previously been ignored. They find that relaxed is defective in excitation–contraction
coupling, whereas sop lacks nicotinic acetylcholine receptors on muscles.
49. Gompel N, Dambly-Chaudiere C, Ghysen A: Neuronal differences
prefigure somatotopy in the zebrafish lateral line. Development
2001, 128:387-393.
50. Hale ME, Ritter DA, Fetcho JR: A confocal study of spinal
interneurons in living larval zebrafish. J Comp Neurol 2001, 437:1-16.
Note added in proof
The work referred to in the text as (LD Hutson, C-B Chien, unpublished data)
is now in press:
51. Hutson LD, Chien C-B: Pathfinding and error correction by retinal
•• axons: the role of astray/robo2. Neuron 2002, in press.
Timelapse imaging and fixed tissue analysis of retinal growth cones in both
wild-type and ast mutants reveials that Astray/Robo2, rather than regulating
midline crossing as in Drosophila, shapes the retinotectal pathway through
the ventral diencephalon. It does so by both preventing and correcting errors
that arise at a low rate during normal embryonic development.