RESEARCH ARTICLE
323
Development 135, 323-332 (2008) doi:10.1242/dev.007559
Position fine-tuning of caudal primary motoneurons in the
zebrafish spinal cord
Mika Sato-Maeda, Masuo Obinata and Wataru Shoji*
In zebrafish embryos, each myotome is typically innervated by three primary motoneurons (PMNs): the caudal primary (CaP), middle
primary (MiP) and rostral primary (RoP). PMN axons first exit the spinal cord through a single exit point located at the midpoint of
the overlying somite, which is formed beneath the CaP cell body and is pioneered by the CaP axon. However, the placement of CaP
cell bodies with respect to corresponding somites is poorly understood. Here, we determined the early events in CaP cell positioning
using neuropilin 1a (nrp1a):gfp transgenic embryos in which CaPs were specifically labeled with GFP. CaP cell bodies first exhibit an
irregular pattern in presence of newly formed corresponding somites and then migrate to achieve their proper positions by
axonogenesis stages. CaPs are generated in excess compared with the number of somites, and two CaPs often overlap at the same
position through this process. Next, we showed that CaP cell bodies remain in the initial irregular positions after knockdown of
Neuropilin1a, a component of the class III semaphorin receptor. Irregular CaP position frequently results in aberrant double exit
points of motor axons, and secondary motor axons form aberrant exit points following CaP axons. Its expression pattern suggests
that sema3ab regulates the CaP position. Indeed, irregular CaP positions and exit points are induced by Sema3ab knockdown,
whose ectopic expression can alter the position of CaP cell bodies. Results suggest that Semaphorin-Neuropilin signaling plays an
important role in position fine-tuning of CaP cell bodies to ensure proper exit points of motor axons.
INTRODUCTION
In a developing nervous system, the final neuronal pattern is often
accomplished by fine-tuning the initial ‘rough sketch’ pattern. For
example, initial connective patterns of some neurons are refined into
an appropriate subset of targets in the final step of determining the
specificity of axonal connections (Purves and Lichtman, 1980;
Nakamura and O’Leary, 1989; Lichtman and Colman, 2000; Kantor
and Kolodkin, 2003). Such a process can provide a mechanism that
ensures appropriate and complete formation of a neuronal pattern.
However, because the process of fine-tuning in the nervous system
has been studied mainly with regard to axonal pattern formation, the
question of whether such a mechanism is applicable to other
neuronal patterns, such as iterative positioning of neuronal elements,
remains unsolved. We have investigated how a regularly spaced
pattern of zebrafish primary motoneuron cell bodies is accomplished
in the spinal cord and examined whether a fine-tuning process is also
involved in the formation of the iterative pattern.
In zebrafish embryos, each myotome is typically innervated by
three identifiable primary motoneurons (PMNs): the caudal primary
(CaP), middle primary (MiP) and rostral primary (RoP) (Eisen et al.,
1986; Myers et al., 1986; Westerfield et al., 1986). Their axons first
exit the spinal cord from a single exit point adjacent to the medial
surface of the somite; the exit point lies in close proximity to the CaP
cell body and is pioneered by the CaP growth cone. All PMN axons
migrate ventrally on the medial surface of the dorsal somite until
they reach the horizontal myoseptal region. At the end of the dorsal
somite pathway, the growth cones encounter a group of specialized
cells called muscle pioneers and then follow divergent pathways that
Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku
University, Sendai 980-8575, Japan.
*Author for correspondence (e-mail: [email protected])
Accepted 8 October 2007
extend to the ventral, dorsal and horizontal myoseptal muscles
within the myotomes (Eisen et al., 1986; Beattie, 2000). CaP axons
start migrating in the numerical order of the segments, and CaP cell
bodies show an iterant regularly spaced pattern (Fig. 1F). Some
spinal hemisegments have two CaP cells; one of which is referred to
as the variably primary (VaP). The two cells are equivalent to each
other morphologically and in gene expression, but one dies about
36 hours after fertilization (Eisen et al., 1990; Eisen and Melancon,
2001). The presence of VaP is different from other PMNs that show
a segmentally arranged pattern; the distribution of VaPs is different
for each embryo and does not always show a bilaterally symmetrical
pattern (Eisen et al., 1990). CaP and VaP cells are positioned in close
proximity and their axons exit from the spinal cord at the same exit
point. The axonal exit points from the spinal cord are also used by
axons of secondary motoneurons (SMNs) that develop later and
follow PMN axons (Westerfield et al., 1986).
The mechanism of zebrafish PMN axonal migration has been
studied extensively (Eisen et al., 1989; Melancon et al., 1997;
Beattie and Eisen, 1997; Zeller and Granato, 1999; Beattie et al.,
2000; Zhang and Granato, 2000; Rodino-Klapac and Beattie, 2004)
and several molecules that function in axon guidance have also been
identified. These include Netrin 1a (Lauderdale et al., 1997),
Semaphorins (Roos et al., 1999; Halloran et al., 2000; Sato-Maeda
et al., 2006), Tenascin C (Schweitzer et al., 2005), and LH3
(Schneider and Granato, 2006). Some of these molecules are
distributed differently within the somite, and it may be necessary for
the PMN axon to decide axonal exit points at the appropriate
positions. However, little is known about the mechanism responsible
for determining the positions of exit points. Although the position
of an exit point may depend on the CaP cell body, the mechanism
responsible for proper positioning of CaP cell bodies corresponding
to the somites is unclear.
Here, we used the sensitivity of GFP expression in neuropilin 1a
(nrp1a):gfp transgenic fish (Sato-Maeda et al., 2006) to trace CaP
cell bodies, and examined early events establishing the iterative
DEVELOPMENT
KEY WORDS: Cell migration, Motoneuron, Neuropilin, Zebrafish
324
RESEARCH ARTICLE
Development 135 (2)
Fig. 1. The pattern of CaP cell bodies before
and after axonogenesis. (A) Optical sections at
different focal levels of an nrp1a:gfp transgenic
embryo at the 28-somite stage (23 hpf). CaPs are
labeled with anti-GFP antibody. Position of CaP cell
bodies was not related to the corresponding
somites. Also, two distinct and discrete cell bodies
were observed at the 25th somite level. Arrows
(lower panel) indicate the somite boundaries.
(B) Irregular patterns of CaP cell bodies were also
observed as the pattern of isl2-expressing cells
(stars) during the pre-axonogenesis period in wildtype embryos. Two separate CaP cells were observed
at the 11th somite level. (C) Appearance ratio of
spinal hemisegments with two discrete CaPs. The
two discrete CaPs were only seen in caudal
segments where CaPs did not begin axonogenesis.
Position 0 is defined as the most caudal segment in
which a CaP axon was formed. Thirty-three pairs of
separate CaPs from 26 embryos at the 26- to 29somite stages were sorted by relative somite levels
and scored for spinal hemisegments with two
separated CaPs. (D) A transgenic embryo at the 29somite stage (23.5 hpf). Although CaP cell bodies
with axons were ellipsoid (20th, 21st), more caudal
CaP cells appeared triangular or trapezoid (23rd,
24th). (E) Side view of a transgenic embryo at the
27-somite stage (22.5 hpf). (F) CaP cell bodies with axons were regularly spaced in the middle of overlying somites. Stars and triangles show
mAb Sv2-labeled CaP/VaP cell bodies. Blue lines denote somite borders. Unless noted otherwise, embryos are oriented as rostral to the left and
dorsal up. Sc, spinal cord; Nc, notochord. The numbers in the panels indicate the segmental order. Scale bars: 50 mm.
MATERIALS AND METHODS
Fish colony
Zebrafish (Danio rerio) were maintained in a laboratory breeding colony at
28.5°C on a 14 hours:10 hours light:dark cycle. Embryos collected from
breeding fish were allowed to develop at 28.5°C and were staged as hours
post fertilization (hpf) and by the number of somites (somite stage) (Kimmel
et al., 1995).
RNA in situ hybridization and immunohistochemistry
Digoxigenin-labeled riboprobes were synthesized by in vitro transcription
and hydrolyzed by limited alkaline hydrolysis (Cox et al., 1984). The
procedure for whole-mount in situ hybridization was described by SchulteMerker et al. (Schulte-Merker et al., 1992). Double labeling in situ
hybridization was performed as described by Whitlock and Westerfield
(Whitlock and Westerfield, 2000). Whole-mount immunostaining was
performed according to the procedures described previously (Shoji et al.,
1998). The primary antibodies were used at the following dilutions: 1/50
for Sv2 (DSHB, University of Iowa), 1/50 for Znp1 (DSHB, University of
Iowa), 1/10 for Zn5/Zn8 (DSHB, University of Iowa), 1/10 for anti-Myc
mAb (Evan et al., 1985), and 1/400 for polyclonal anti-GFP antibody
(Invitrogen). To label PMN axons at 48 hpf, we used mAb Znp1 instead
of Sv2 because of its greater sensitivity. For immunostaining with in situ
hybridization, fixed embryos were first processed for immunostaining
followed by re-fixation and processing for in situ hybridization. Semi-thin
sections were cut with a microslicer (Dosaka EM, Kyoto, Japan) after
whole-mount hybridization or immunostaining according to the
procedures describe previously (Sato-Meda et al., 2006).
Detection of CaP cell bodies
In addition to the specific molecular marker isl2 for CaPs (Appel et al.,
1995; Tokumoto et al., 1995), we used a monoclonal antibody Sv2 and
the nrp1a:gfp transgenic strain to identify CaP cell bodies. Sv2 was
originally established as a monoclonal antibody that recognizes
transmembrane glycoprotein in synaptic vesicles (Buckley and Kelly,
1985) and was reported to label all primary motor axons in zebrafish
embryos (Panzer et al., 2005; Schneider and Granato, 2006). Instead, we
found that Sv2 labeled the cell body and axon of CaPs during early
axonogenesis in embryos earlier than 24 hpf (30-somite stage). In
embryos at this stage, isl1-positive neurons in the ventral spinal cord that
correspond to MiP and RoP showed no immunostaining with Sv2 (Fig.
3F). Therefore, we determined the Sv2-labeled neurons to be CaPs, and
not MiPs or RoPs, at this early stage of axonogenesis. For fluorescent live
images, GFP-labeled cell bodies were examined with a Zeiss Axioskop
upright microscope or with an Axiovert LSM5 Pascal confocal
microscope.
CaP position irregularities were defined as follows. For pre-axonogenesis
stages, CaPs situated on or by the borders of overlying somites at caudal five
levels in 25- to 30-somite stages embryos. For stages after axonogenesis,
CaPs located at the anterior or posterior marginal quarters (Fig. 3E) on the
focus of the dorsal edge of the notochord.
Prediction of axonal extension according to segment and
developmental stage
We determined the average status of CaP axonal development in segments
at each stage (Sato-Maeda et al., 2006). This information enabled us to
determine approximate segments in which the CaP axons begins
axonogenesis at a given developmental stages (Table 1). Axons were
detected using mAb Znp1.
DEVELOPMENT
pattern. The regularly spaced pattern of CaP cell bodies was not
present initially, but was achieved gradually by the time of
axonogenesis. VaP was formed by spatial fine-tuning of CaP cell
bodies, present in excess relative to the somites. This position
adjustment was disrupted by Nrp1a knockdown, which often
resulted in aberrant axonal exit points. Correspondingly, irregular
patterns of CaPs were observed in Sema3ab knockdown embryos,
and CaP cell bodies showed repulsive reaction to ectopic Sema3ab.
These results suggest that the Semaphorin-Neuropilin signal plays
an important role in fine-tuning the CaP position, which ensures
proper exit points of axons and harmonizes development of spinal
motoneurons and segmented somites.
Position fine-tuning of motoneurons
RESEARCH ARTICLE
325
Table 1. Segment range where CaP axons begin axonogenesis at each somite stage
Somite stage
Embryos examined
Segment position*
Segment range
22
9
14.4±0.88
13-16
23
15
15.2±0.56
14-16
24
11
15.8±0.98
14-17
25
10
17.1±0.88
16-18
26
11
18.6±1.03
17-20
27
16
19.4±0.81
18-21
28
11
19.7±1.10
18-21
29
12
22.3±0.65
21-23
Morpholino oligonucleotide injection
The antisense morpholinos (25-mer) were designed against DNA sequences
for 5⬘-UTR or splicing donor sites (indicated as ‘a splice blocker’). The
effectiveness of sema3aa MO, sema3ab MO1 and nrp1a MO1 was reported
previously (Shoji et al., 1998; Lee et al., 2002; Torres-Vazquez et al., 2004).
The control morpholino sequence had a five-base mismatch. The sequences
were as follows:
sema3aa MO: 5⬘-CTTGTAGCCCACAGTGCCCAGAGCA-3⬘;
sema3aa MO control: 5⬘-CTTCTAGCCGACAGAGCCCAGTGCA-3⬘;
sema3ab MO1 (a splice blocker): 5⬘-AAATGTGTCTTACCGTTGAGCCATC-3⬘;
sema3ab MO1 control: 5⬘- AAATCTGTGTTACGGTTCAGCGATC-3⬘;
sema3ab MO2: 5⬘-GTTCCGTATGCAGTCCCGTGGCCTC-3⬘;
sema3ab MO2 control: 5⬘-GTTCGGTATCCACTCCCCTGGACTC-3⬘;
nrp1a MO1: 5⬘-GAATCCTGGAGTTCGGAGTGCGGAA-3⬘;
nrp1a MO1 control: 5⬘-GAATGCTCGACTTCGGAGTCCGCAA-3⬘;
nrp1a MO2 (a splice blocker): 5⬘-GCTCAACACTCACTTGCACTCTCGG-3⬘; and
nrp1a MO2 control: 5⬘-GCTGAAGACTCAGTTGCAGTCTGGG-3⬘.
MOs were solubilized in 1⫻ Danieau Solution (Nasevicius and Ekker,
2000) and were injected into recently fertilized eggs (approximately 6-9 ng
for each embryo). To obtain 48 hpf Nrp1a knockdown embryos, diluted
nrp1a MO1 (2-3 ng/embryo) was injected.
DNA injection
Approximately 1 nl of a 50 ng/ml solution of hsp70:sema3ab-myc or
hsp70:myc in water containing 0.1% Phenol Red was pressure injected
from a micropipette into recently fertilized eggs as described previously
(Sato-Maeda et al., 2006). To induce expression, embryos were incubated
at 38°C for 30 minutes at the 22-somite stage. After heat induction,
embryos were cultured until the 30-somite stage (24 hpf) and
immunostained with Sv2 and anti-Myc or anti-GFP. Segments in which
the construct was integrated into the floor plate cells and posterior to the
18th segment were examined.
RESULTS
CaP position is adjusted gradually before axon
formation
The distribution of CaP cell bodies changes during development.
The expression pattern of zebrafish isl2, a molecular marker for
CaPs, did not correspond to the somite arrangement in the early
spinal cord at the 8-somite stage when looking at the sixth to seventh
somite level (Appel et al., 1995). By contrast, the CaP cell bodies at
the axonogenesis stage are regularly spaced at the midportion of
overlying somites (Eisen et al., 1986) (Fig. 1F, 17-20th segments at
29-somite stage; Fig. 3E, scheme). The difference suggests that the
position of CaP cell bodies is regulated with the segmented somite.
To examine when and how the position is established, we used
nrp1a:gfp transgenic embryos as previously described (Sato-Maeda
et al., 2006). In this transgenic strain, GFP is expressed by CaPs and
VaPs according to endogenous nrp1a expression (Feldner et al.,
2005; Sato-Maeda et al., 2006). Our series of live observations
determined that CaPs and VaPs solely express GFP during the preaxonogenesis stage in transgenic embryos (n=40). However, other
motoneurons and interneurons in the ventral spinal cord begin
expressing GFP after 24 hpf, which made longer tracing difficult
(Sato-Maeda et al., 2006).
GFP-expressing CaPs were first detected when the corresponding
somite was newly segmented. At younger somite levels, where the
CaPs had not yet begun axonogenesis, the cell bodies were
irregularly distributed (Fig. 1A and Table 1; 24-27th segments at 28somite stage). Some cell bodies were situated in the anterior or
posterior portion; others were situated on the boundary of the
overlying somite. In addition, two CaP cell bodies were often
observed within the region of a single somitic segment (cells in the
25th segment in Fig. 1A), which were also observed by isl2
expression (Fig. 1B: 11th segment at 17-somite stage). Severe
displacement of CaP cell bodies on or by the border of the overlying
somite was seen in 52% of the spinal segments (34 out of 65), in
which 35% and 17% had one and two CaPs, respectively. The two
CaPs discretely located under a somitic segment were seen only
during the pre-axonogenesis period (Fig. 1C) and merged at the
midportion after that period. This time course indicates that the CaP
position is established by the time of axonogenesis.
CaP cell bodies altered their appearance dynamically (Fig.
1A,D,E). At first, they appeared flat, clinging to the floor plate (26th
and 27th segments in Fig. 1A). In more rostral (i.e. older) levels, they
were triangular or trapezoidal (23rd and 24th segments in Fig. 1D).
When CaP started to form axons, they were all ellipsoidal (20th and
21st segments in Fig. 1D). Transition of the CaP cell shape was more
clearly demonstrated in live observations using nrp1a:gfp transgenic
embryos (Fig. 2). Here, we were able to identify GFP-labeled CaP
cells that altered their appearance from flat to triangular (Fig. 2A
star, Fig. 2B).
The irregular initial pattern of CaPs was adjusted to the regularly
spaced pattern by active cellular movement. When we followed
GFP-positive cells at the newly forming somite level, a flat-shaped
CaP originating under the somite boundary migrated posteriorly to
the midsection (arrowhead in Fig. 2B) and it eventually overlapped
with another CaP. Then, the two equivalent neurons were distributed
under the midportion of a corresponding somite, on being the CaP
and the other the VaP. Similar movement was frequently seen in
which two discrete cells overlapped 80 minutes later (arrowhead in
Fig. 2A; the 21st segment at the 27-somite stage in the top panel).
As mentioned above, these two separate CaP cells were only seen at
younger (caudal) somite levels when CaP axons were not formed
(Fig. 1C), which indicates that migration is completed before
axonogenesis.
Our observations showed that the regularly spaced pattern of CaP
cell bodies was achieved by cellular migration. They were
distributed initially in an irregular pattern and then adjusted their
position to correspond with the somite by the time of axonogenesis.
This process appears to be responsible for the heterogeneity in the
spinal segments; some have only a CaP, whereas the others have a
CaP and a VaP (Fig. 1F).
Antisense knockdown of nrp1a results in
abnormal CaP position even after axon formation
Our observations indicated that the position fine-tuning of the CaP
cell body occurs prior to axonogenesis. To identify the molecules
involved, we studied the effects of Nrp1a knockdown on the CaP
DEVELOPMENT
*Average position (segment number) of the most caudal segment with a CaP axon, ± s.d.
326
RESEARCH ARTICLE
Development 135 (2)
Fig. 2. Live observations of CaP cell bodies in nrp1a:gfp
transgenic embryos. (A) Trunk region of the same embryo shown at
different stages. Two discrete cells underlying a single somite seen in
the upper panel (the 21st somite level at the 27-somite stage:
arrowhead) overlapped after 80 minutes, shown in the lower panel.
A transition in cell shape between different stages was also observed
(stars). The numbers in the panels indicate the segmental order. Scale
bar: 50 ␮m. (B) Confocal micrographs of CaP cell bodies in another
embryo. Sequential images were captured of the 16-18th segment,
starting from the 17-somite stage (17.5 hpf). A CaP cell body originally
located beneath the somite boundary (arrowhead) migrated posteriorly
and overlapped with another CaP in the middle of the somite. The
numbers denote the time elapsed. The lines indicate segment borders
of the somites. Scale bar: 20 ␮m.
position. nrp1a is expressed by CaPs when fine-tuning their
position, and knockdown of Nrp1a has reportedly brought about
various degrees of aberrant axonal phenotype (Feldner et al., 2005).
To determine whether Nrp1a is required for CaP cell positioning, we
injected antisense MOs against nrp1a into recently fertilized
embryos and assayed the CaP cell position with isl2 in situ
hybridization and with Sv2 immunostaining.
In Nrp1a knockdown embryos, CaP cell bodies initially arose in
an irregular pattern similar to that in wild-type embryos (see Fig. S1
in the supplementary material). However, CaPs were out of normal
alignment when the position became regularly spaced in intact and
control MO-injected embryos (Fig. 3A-D). Significant numbers of
CaPs were dislocated severely at the marginal quarters to the
corresponding somite (Fig. 3E) compared with the control.
Moreover, two separate CaP cell bodies did not merge (Fig. 3B,D),
and their axons left the spinal cord independently. These two discrete
exit points were the most notable phenotype in Nrp1a knockdown
embryos (Fig. 3F,G; see Table 3), which was associated with the
position fine-tuning defect.
Because of these defects in the positions of CaPs, we assumed that
the aberrantly positioned cells would be subjected to different
environments that may affect the subtype identity of the CaP.
Knockdown of Sema3ab also results in abnormal
CaP position
We next investigated class III semaphorins, ligands of Nrp1a, which
would function in the position fine-tuning of CaP cell bodies. Two
copies of the zebrafish sema3a genes, sema3aa and sema3ab
(previously called sema3a1 and sema3a2, respectively) (Yee et al.,
1999; Roos et al., 1999), were potential candidates because they
were expressed in myotomes. However, the level of sema3aa
expression was much weaker compared with sema3ab during the
CaP pre-axonogenesis period (Shoji et al., 1998; Yee et al., 1999;
Bernhardt et al., 1998). First, we examined the effects of the
knockdown of these genes by screening the double exit phenotype
of CaP axons, which indicates displacement of CaP cell bodies.
Injection of sema3aa MO caused few aberrant exit phenotypes
(Table 3). These phenotypes were mild, with two cell bodies
overlapped slightly (data not shown). By contrast, knockdown of
Sema3ab frequently caused double exit phenotype of axons with
irregular distribution of the CaP cell bodies (Fig. 4A,B; Table 3).
Interestingly, the extent of the effect was less than that with Nrp1a
knockdown, which may indicate that there would be more ligands
involved with Sema3ab. However, Sema3aa is not likely to play this
role with Sema3ab, because the defect with Sema3aa/Sema3ab
double knockdown was not greater than that with the single
Sema3ab knockdown. By contrast, another set of double
knockdowns for Nrp1a and Sema3ab resulted in an added effect on
the double exit phenotype compared with each single knockdown
(Table 3), which is consistent with the idea that the products of these
genes work together as a ligand-receptor complex. Alternatively, this
additive effect would suggest Nrp1b, which is weakly expressed
by CaPs (M.S.-M., unpublished observation), may partially
compensate for the CaP positioning. Thus, Semaphorin-Neuropilin
signaling is considered necessary for the proper positioning of CaP
cell bodies, and Sema3ab functions as a ligand of Nrp1a in this
process.
The expression pattern of sema3ab in zebrafish was reported
previously (Bernhardt et al., 1998; Roos et al., 1999; Shoji et al.,
2003); its expression is detected from 12 hpf in homogeneous
unsegmented mesoderm. As the somite develops to the segmented
form, the striped expression pattern emerges as it localizes to the
posterior half of each somite (Bernhardt et al., 1998). Here, we
DEVELOPMENT
However, molecular marker analysis indicated that this is unlikely
because both of the separated cells expressed isl2, not isl1, markers
of MiP and RoP cells (Fig. 3F,G). In addition, isl2+ cell number in
the ventral spinal cord throughout a given segmental level did not
change between Nrp1a knockdown and the control embryos (Table
2). These results suggest that differentiation and subtype
specification of CaP were unaffected in the Nrp1a-knockdown
embryos. Furthermore, axons of the two separated CaPs, caused by
Nrp1a knockdown, had features similar to those of a CaP and a VaP;
only one CaP can extend its axon to the ventral myotome, and the
other cell (i.e. VaP) ceases axonal extension at the horizontal
myoseptal region (Eisen et al., 1990; Eisen and Melancon, 2001).
As shown in Fig. 3G and Fig. 7A, only one axon migrated further
onto the ventral myotome, and the other axon did not migrate
beyond the horizontal myoseptal region in most cases (26 of 29 pairs
of axons).
These results suggest that Nrp1a is required for position finetuning of CaP cell bodies, and that the process occurs after
differentiation and subtype specification of the CaP. Defects in the
fine-tuning result in aberrant exit points due to the separation of CaP
and VaP cells.
Position fine-tuning of motoneurons
RESEARCH ARTICLE
327
Fig. 3. Knockdown of Nrp1a results in abnormal CaP cell
positioning even after axon formation. Position of CaP
cell bodies after axonogenesis was examined using isl2
expression (purple) and mAb Sv2 (brown). (A) Side view of a
control embryo at the 28-somite stage. (B) A Nrp1aknockdown embryo at the 29-somite stage with an irregular
CaP pattern. (C,D) Horizontal sections of control and Nrp1aknockdown embryos. CaPs are indicated by stars. (E; left)
Severe dislocation was defined as the center of the CaP cell
bodies being located in either the anterior or the posterior
quarter of the overlying somite (indicated by ‘Mar’ in the
schema). (Right) Dislocation of CaP was significantly
increased in Nrp1a-knockdown embryos, as determined by
Fisher’s exact test of independence (star, P<0.05).Nc,
notochord. (F) Abnormal exit points of CaP axons by two
separate CaPs in Nrp1a-knockdown embryos. CaP axons
were labeled with mAb Sv2. (G) The positions of CaPs (stars)
relative to MiP and RoP (diamonds, labeled by isl1) were
unaffected by Nrp1a knockdown. (H) Different lengths of
two axons from separate CaPs correspond to those of a CaP
and a VaP. One axon extended onto the ventral myotome (*),
whereas the other axon did not extend beyond the horizontal
myoseptum (**). Arrowhead shows the level of the
horizontal myoseptum. MiP axons extended normally along
the dorsal pathway (arrows). The numbers in A, B and F-H
denote the somitic segment order. The blue lines indicate
segment borders. Scale bar: 50 ␮m.
CaP cell bodies are repulsed by cells
misexpressing Sema3ab
The nrp1a and sema3ab expression patterns and results of
knockdown studies suggest that Sema3ab may repulse CaP
cell bodies. Roos et al. (Roos et al., 1999) reported that the CaP
axons stalled in embryos injected with sema3ab mRNA, and the
authors suggested that Sema3ab can repulse CaP axons. To
determine whether Sema3ab can repel CaP cell bodies as well as
their axons, we injected embryos with DNA constructs of
Table 2. Number of islet 2(+) primary motoneurons in nrp1a
embryos injected with morpholino oligonucleotides (MOs)
Somite stage
16-18
20-23
Segments*
MO
Embryos
examined
Mean ± s.d.
10 segments
(4th-13th)
10 segments
(8th-17th)
nrp1a
Control
nrp1a
Control
16
16
17
19
12.56±1.26
13.31±0.95
13.24±1.03
13.84±0.83
冎
冎
†
†
*The number of islet 2(+) cells throughout 10 somitic segments was scored. Either
side of the 4th-13th or 8th-17th segments were examined at the 16-18 or 20-23
somite stages, respectively.
†
No significant difference at a significance level of 0.05 (Student’s t-test).
hsp70:sema3ab-myc or hsp70:myc. In these embryos, a random
mosaic of cells expressed exogenous Sema3ab after heat
induction.
When CaP cell bodies encountered floor plate cells expressing
focal ectopic Sema3ab, they shifted anteriorly or slightly dorsally
away from the Sema3ab-expressing cells (nine out of nine CaPs
Table 3. Knockdown of Nrp1a and Sema3ab causes abnormal
axonal exit points
MO injected (ng/embryo)
nrp1a MO1 (7)
nrp1a MO1 control (7)
nrp1a MO1 (3)‡
nrp1a MO2 (5)
nrp1a MO2 control (5)
sema3aa MO (9)
sema3aa MO control (9)
sema3ab MO1 (9)
sema3ab MO1 control (9)
sema3ab MO2 (3)
sema3ab MO2 control (3)
sema3aa MO (7) + sema3ab MO1 (7)
nrp1a MO1 (7) + sema3ab MO1 (7)
Embryos
examined
49
42
30
45
42
39
39
41
51
46
53
31
33
Embryos with double
exit points (%)
冎*
15
0
5
10
1
2
0
6
1
4
0
5
12
冎*
冎
冎*
冎*
†
(30.6)
(0)
(16.7)
(22.2)
(2.4)
(5.1)
(0)
(14.6)
(2.0)
(8.7)
(0)
(16.1)
(36.4)
Injected embryos were fixed at the 25-29 somite stage.
*By Fisher’s exact test of independence, morpholino oligonucleotide (MO) injection
was considered effective at producing the double exit phenotype (P<0.05).
†
Effect of the MO injection was not significant. The possibility that the double exit
phenotype and MO injection might have been independent events cannot be
rejected at a 0.05 significance level.
‡
Less MO was applied to the embryos shown in Fig. 6 to circumvent embryonic
death by circulation defects so as to enable examination of secondary motoneuron
axons.
DEVELOPMENT
further examined the expression in relation to the positions of CaP
cell bodies during the CaP pre-axonogenesis period. Expression of
sema3ab was most noticeable in somites in which the corresponding
CaP cell bodies were being adjusted (19th-21st; Fig. 4C). In these
somites, CaPs were situated under the margin of the expressing
region, which suggests that CaP cell bodies may determine their
position when they detect a particular level of Sema3ab in their
environment.
RESEARCH ARTICLE
Fig. 4. Knockdown of Sema3ab causes the double exit phenotype
of CaP axons similar to Nrp1a knockdown. (A) The position of CaP
cell bodies after axonogenesis was examined using isl2 expression.
Separate isl2+ CaP cell bodies were detected in Sema3ab-knockdown
embryos (11th segment of a 25-somite stage embryo). (B) The double
exit phenotype of CaP axons was observed in a Sema3ab-knockdown
embryo. CaP axons and cell bodies were labeled with mAb Sv2.
(C) sema3ab was expressed in the posterior halves of somites, and CaP
cell bodies (detected by isl2 expression) were observed around the
margin of the sema3ab expressing region. The inset shows the 21st
segment of the same embryo at the focal plane of the CaP cell bodies.
In older segments in which CaP axonogenesis had begun, expression of
sema3ab was downregulated (17th segment of a 25-somite stage
embryo). The numbers indicate the segmental order, and the stars
indicate the positions of the CaP cell bodies. Scale bar: 50 ␮m.
were mislocated; Fig. 5A,B). In controls, CaP cell bodies were all at
the normal position and overlapped with cells expressing the Myc
epitope (four out of four CaPs; Fig. 5C,D). These results suggest that
Sema3ab regulates the position of CaPs in a repulsive manner, to
achieve position fine-tuning.
Irregular CaP position causes aberrant exit points
of SMN axons
We examined whether abnormal CaP exit points would affect
following axons. In the zebrafish spinal cord, axons of SMNs
recognized by monoclonal antibody Zn5 (black axons in Fig. 6A)
follow the pathway of PMN axons (brown axons in Fig. 6A) and use
a common and narrow exit region in the midportion. Pike et al. (Pike
et al., 1992) reported that SMN ventral axons extended slowly and
form aberrant branches after ablation of CaP, but were still able to
reach ventral myotomes. This study suggested that CaP axons
facilitated SMN axonal development, but it is still unclear how exit
points of SMN axons are determined. Here, we examined SMN
axons in Nrp1a-knockdown embryos in which double exit points of
PMN were induced.
In Nrp1a-knockdown embryos, two separate SMN exit points
adjacent to a single somite were observed (four segments in two
out of 21 embryos; Fig. 6B,C). By contrast, we did not observe
Development 135 (2)
Fig. 5. Abnormal CaP cell positioning induced by focal ectopic
Sema3ab. (A,B) Optical sections at different focal levels of an embryo
in which Sema3ab-Myc was transiently expressed. Ectopic Sema3ab
was labeled with anti-Myc (black) and CaPs with mAb Sv2 (brown). In
a case in which ectopic Sema3ab was expressed in floor plate cells
(white double arrows in A), the CaP cell body was shifted anteriorly
(arrowhead in B) away from the cells expressing Sema3ab.
(C,D) Optical sections of a control embryo. Myc epitope expression
(white double arrows in C) did not affect the location of the CaP cell
body (arrowhead in D) and overlapped with it. Lines in B and D
indicate somitic borders. Sc, spinal cord; FP, floor plate; Nc, notochord.
Scale bar: 50 ␮m.
aberrant SMN exit points in relation to the PMN axon in the
control embryos (19 embryos). The defects in SMN axons were
relatively moderate and less frequent, as correlated with the lesser
amount of MO applied in the experiment, but were consistent with
the frequency of CaP axon defects induced by the same dose
(Table 3). Although nrp1a MO induced strong double exit
phenotype and resulted in embryonic death by 48 hpf, probably
because of circulation defects (Lee et al., 2002), it was necessary
to reduce the amount of MO injected. Even at this less effective
dose, these double exit points induced the formation of two
bundles of SMN axons; one bundle was thicker and clearly lay
alongside the PMN axons (Fig. 6B,C, arrowheads). The other
bundle, some located anteriorly and others posteriorly, was
thinner and sometimes loose (Fig. 6B,C, arrows). One of the two
bundles did not associate with the PMN axon, which probably
mirrors VaP axons that would degenerate at the stage examined.
We further examined the correlation between CaP and SMN
defects by tracking the double exit points of CaP using nrp:gfp
transgenic embryos with Nrp1a knockdown. When we found the
double exit points of CaP axons at 24-25 hpf, the double exit
phenotype in SMN axons appeared at 48 hpf in most cases (eight
out of 11 cases: Fig. 7). In several remnant cases, separated CaPs
formed double exit points once; however, two axons merged near
the exit points after sometime, resulting in a SMN abnormality,
which was difficult to observe.
These results indicate that SMN exit points are dependent on the
PMN exit points. Thus, the position fine-tuning of CaP is also
important for SMN axon development. If the fine-tuning is not
correct, subsequent SMN axons behave abnormally, thereby
disrupting the coupled segmental architecture between the spinal
cord and segmented somites.
DEVELOPMENT
328
Fig. 6. Aberrant exit points of secondary motoneuron (SMN)
axons in Nrp1a-knockdown embryos. (A) Axons of primary
motoneurons (PMNs) and SMNs at 48 hpf in a control MO-injected
embryo. PMN axons were labeled with a brown stain, recognized by
mAb Znp1, but not by Zn5 (Zeller et al., 2002). SMN axons were
stained in blue-black, recognized by Zn5 (Fashena and Westerfield,
1999), regardless of Znp1 reactivity. SMN axons extended ventrally
along with PMN axons using an exit point common to PMN and SMN.
(B) Double exit phenotype of SMN axons was observed in Nrp1aknockdown embryos at 48 hpf. Generally, one axonal bundle of SMNs
was thicker (arrowhead) than the others (arrow), regardless of its
anterior or posterior position. (C) Another example of a double exit
phenotype of SMN. Scale bar: 50 ␮m.
DISCUSSION
Spatial and numerical fine-tuning of CaP cell
bodies
In the present study, we investigated how CaP cell bodies form a
regularly spaced iterative pattern in the zebrafish spinal cord. Our
results showed that the cell bodies are distributed irregularly at first
and do not correspond precisely to newly forming somitic segments
(Fig. 8A,B). After the neighboring paraxial mesoderm is
segmented, the CaP cell body migrates anteriorly or posteriorly to
adjust its position corresponding to the middle area of the overlying
somite (Fig. 8C, left). This fine-tuning of the CaP position ensures
regularly spaced axonal exit points beneath the cell bodies, which
are later followed by SMNs. This ‘spatial fine-tuning’ is required
to establish segmental architecture of the spinal motor nerve and
somite, in which each segmental nerve bundle passes through
iterative vertebrae and innervates the same level of myotomes. In
cases where the CaP position was not properly adjusted (Fig. 8C,
right), the common exit points were abnormally localized (Fig. 8D,
right). Although the reason why CaP axons, but not secondary
motor axons, exit the spinal cord beneath their cell bodies remains
unclear, our results indicate that the CaP position determines the
common exit points. The spatial fine-tuning of CaP cell bodies is
thus important for harmonizing the spinal cord and somite
development.
Along with the irregular position, the initial cell number of CaPs
exceeds the number of somites (Fig. 8B,C, left; Table 2), which is
necessary to provide at least one cell under each somitic segment
during the tuning process. The excessive CaPs also result in two
CaPs along corresponding somitic segments, although they do not
disrupt the one exit point per somitic segment rule because the two
CaPs overlap at their proper position through spatial fine-tuning
(Fig. 8D, left). Subsequently, only one CaP survives and the other is
eliminated as a VaP (Beattie et al., 2000; Eisen and Melancon, 2001),
and this ‘numerical fine-tuning’ of CaPs can be achieved without
affecting either the exit points or other segmental structure as spatial
fine-tuning is accomplished successfully (Fig. 8E, left). Neural
systems often adopt an ‘excess innervations and subsequent
elimination’ strategy, i.e. an excessive number of axons are allowed
to connect to a limited number of targets, but eliminated later by
RESEARCH ARTICLE
329
Fig. 7. Abnormal CaP cell positioning results in double exit points
in both primary motoneurons (PMNs) and secondary
motoneurons (SMNs). (A) Sequential images at the 18th somite level
in an nrp1a:gfp transgenic embryo with Nrp1a-knockdown. Two
separated CaP cell bodies (21 hpf) extended their axons to form double
exit points (24 hpf). At 27 hpf, one axon extended beyond the
horizontal myoseptal level (*), but the other axon did not (**).
Arrowheads indicate the level of the horizontal myoseptum. (B) The
double exit phenotype of SMN axons was observed at the same
position as in A at 48 hpf. SMN axons were immunostained with mAb
Zn5. Scale bars: 20 ␮m.
activity and/or trophic support-dependent processes (Oppenheim,
1991; Balice-Gordon and Lichtman, 1994; Katz and Shatz, 1996;
Pettmann and Henderson, 1998). Excessive CaPs and elimination as
VaPs in zebrafish spinal cord can be regarded as a similar type of
numerical fine-tuning. Here, spatial fine-tuning is an important event
for maintaining coupled development between spinal motoneurons
and their target somitic myotomes within the numerical fine-tuning
process.
Fine-tuning and subtype specification of CaP
Although the results of this study indicate the position fine-tuning
of CaPs is in accordance with the segmented somites, several lines
of evidence suggest that the paraxial mesoderm also provides cues
to specify the differentiation of primary motoneurons to CaP, MiP
or RoP in each spinal segment (Eisen, 1991; Lewis and Eisen, 2004).
This raises the possibility that the two processes, i.e. subtype
specification and position fine-tuning, may be interdependent. The
extreme version of this hypothesis has the CaP identity being
established by the cell’s position within the somitic segment. For
example, only cells that migrate and are located at the midportion of
the somite would become CaPs, whereas those that did not reach this
position would differentiate into other types of neurons. However,
this seems unlikely because our real-time observations showed that
nrp1a:gfp-positive neurons that will become CaPs are already
present at the newest somite level in an irregular pattern (Fig. 1A,
Fig. 2B) and that their migration defect does not change molecular
marker expression or ventral projecting feature of the CaP feature
(Fig. 3F-H). The above mentioned evidence indicates that the
subtype identity of the CaP is determined before the somite
establishes morphological boundaries, and the position fine-tuning
occurs subsequent to cell-type specification. The results of other
studies support this sequence. Eisen (Eisen, 1991) reported that CaP
cell bodies transplanted to the MiP positions moved back to the
original positions. In addition, Lewis and Eisen (Lewis and Eisen,
2004) reported that several zebrafish mutants lack morphological
somitic segments but retain early cryptic segmentation as revealed
by her1 and cs131 expression in the presomitic mesoderm. In these
mutants, primary motoneurons are specified as CaPs or MiPs, but
the precise spacing is disturbed. Further studies are needed to
DEVELOPMENT
Position fine-tuning of motoneurons
330
RESEARCH ARTICLE
Development 135 (2)
Fig. 8. Schematic summaries for fine-tuning of CaP cell
positioning to correspond to overlying somitic segments. The
differentiation and subtype specification of CaPs (A), somite
segmentation (B), spatial fine-tuning (C), axonogenesis (D), and
numerical fine-tuning (E). CaP cell bodies initially do not correspond
precisely in position to newly segmented somites (A,B). In wild type, by
the time of axonogenesis, CaP cells are adjusted to the proper positions
in relation to somitic segments (C, left). When two CaPs are overlaid by
a single somite, they overlap their position with each other, thereby
maintaining one axonal exit point per adjacent somite (D, left). Later,
one of the two CaPs is eliminated as VaP and each ventral myotome
comes to be innervated by only one CaP (E, left upper diagram). If the
spatial fine-tuning is successful, secondary motor axons born later
(purple, lower diagram) follow the single exit point in each segment.
However, after knockdown of Nrp1a or Sema3ab, CaP cell bodies
remain in the initial irregular positions (C, right). Abnormal CaP cell
positioning brings about the double exit phenotype of CaP axons (D,
right) and these are followed by secondary motor axons (purple; E,
right lower).
understand the subtype specification of PMNs; however, here we
concluded that the specification as a CaP and its fine-tuning occur
as sequential and separate processes.
Semaphorin-Neuropilin signaling plays an
important role in position fine-tuning
The fine-tuning process is regulated by the Semaphorin-Neuropilin
signal that functions in various types of cell migration and neural
growth cone guidance (Kolodkin, 1998; Raper, 2000; Kruger et al.,
2005). sema3ab, a secreted class III semaphorin gene, is expressed
by newly forming somites (Roos et al., 1999; Shoji et al., 2003),
whereas nrp1a, which encodes a receptor subunit for Sema3A
(Kolodkin et al., 1997; He and Tessier-Lavigne, 1997) is expressed
specifically by CaPs (Sato-Maeda et al., 2006). Knockdown of each
gene resulted in similar position defects in CaP cell bodies,
consistent with the hypothesis that the products of these genes work
together as a ligand-receptor complex to determine the CaP position.
In analogy with the repulsive action of Sema3A on growth cone
Semaphorin-Neuropilin signal functions in cell
migration and axon guidance of CaP
The results presented herein along with those of our previous study
(Sato-Maeda et al., 2006) suggest that two copies of the zebrafish
sema3a gene function in position fine-tuning and in pathfinding of
CaP axons. First, sema3ab expressed in newly forming somites
regulates the position fine-tuning described above; second, sema3aa
expressed, in turn, in a different pattern controls the navigation of
axon pathfinding (Shoji et al., 1998; Shoji et al., 2003; Sato-Maeda
et al., 2006). Along with this transition, somitic expression of the
sema3a genes changes from the posterior region by sema3ab to the
dorsal and ventral regions, but not in between by sema3aa (Shoji
et al., 1998; Shoji et al., 2003; Sato-Maeda et al., 2006). This
subfunctionalization by two homologous genes (Lynch and Force,
2000) is also supported by our knockdown studies. In Sema3abknockdown embryos, the CaP position is abnormal, whereas the
axon pathfinding appears normal (Fig. 4B). By contrast, Sema3aa
knockdown barely affects the CaP position (Table 3), whereas the
axon behaves abnormally (Sato-Maeda et al., 2006). During position
fine-tuning, Sema3ab regulates migration of the cell body. However,
after axonogenesis, Sems3aa does not regulate the cell body, but
regulates migration of growth cones and axons. Thus, two sema3a
genes regulate the fine-tuning and axon pathfinding processes
sequentially and separately. It remains unclear how these different
events are finely regulated in individual cells. Because the cell
morphology is quite different – flat during cell migration, changing
to triangular, trapezoidal, and finally to ellipsoidal during axon
formation – we speculate that subcellular domains that respond to
semaphorins may be switched between these two processes.
Alternatively, the cell body may be anchored by its adhesive nature
after position tuning to maintain the cell arrangement inside the
spinal cord. The regulatory mechanism controlling which portion of
neuronal cells are motile and which immotile is a profound issue,
and CaP development would be a good model to investigate such
questions in future studies.
In conclusion, a stepwise fine-tuning process accomplishes the
regularly spaced pattern of CaP cell bodies and its relationship to
somitic segments. After subtype specification, the initial ‘rough
DEVELOPMENT
guidance (Luo et al., 1993), we hypothesized that Sema3ab affects
CaP migration in a repulsive manner. In fact, CaP position was
shifted by the presence of nearby ectopic Sema3ab-expressing cells
(Fig. 5). An endogenous sema3ab expression pattern, at the posterior
region of segmented somites (Roos et al., 1999; Shoji et al., 2003)
(Fig. 4C), provides partial support for this idea. In cases where a CaP
faces the posterior of the segmented somite, it would migrate
anteriorly by detecting the Sema3ab level in the environment. In this
model, however, the position fine-tuning cannot be fully explained
in the case in which the CaP faces the anterior of the segment. One
potential explanation for this is that an additional repulsive factor,
such as another semaphorin, may be expressed in the anterior region
of the somite and play a role. Interestingly, the frequency of
abnormal CaP due to Sema3ab knockdown was about half that
caused by Nrp1a knockdown. This result indicates that additional
molecules may be involved as ligands for Nrp1a. Of the nine
members of the class III semaphorins identified in zebrafish
(Halloran et al., 1998; Roos et al., 1999; Yee et al., 1999; Yu et al.,
2004; Stevens and Halloran, 2005), sema3h is expressed at the
anterior region of the segmented somite (Halloran et al., 1998;
Stevens and Halloran, 2005) and is thus a good candidate to support
our model. However, the potential relationship between this novel
semaphorin and CaP migration should be examined in future studies.
sketch’ of the CaP cell pattern is adjusted to a ‘fine pattern’, which
ensures the proper axonal exit point and harmonizes the spinal cord
and somite development. The Semaphorin-Neuropilin signal plays
an important role in this process, although at present it can only be
partially explained. Further studies on other semaphorins as well as
other molecules are required to understand the molecular
mechanism underlying this process.
We thank Drs Halloran and Kuwada for helpful discussions and comments,
and M. Ajiro, H. Tawarayama and L. Li for their technical expertise. This work
was funded by Grant-in Aid for Scientific Research and Dynamics of
Extracellular Environments to W.S. National BioResource Project, Japan,
provided fish lines. Developmental Studies Hybridoma Bank, University of
Iowa, provided antibodies.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/323/DC1
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