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First published online 30 August 2006
doi: 10.1242/dev.02559
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1 Department of Physiology and Biophysics, 8307 University of Colorado Denver
and Health Sciences Center, Aurora, CO 80045, USA.
2 Department of Biological Sciences, Louisiana State University, Baton Rouge, LA
70803, USA.
3 Department of Biological Sciences, Vanderbilt University, Nashville, TN 37232,
USA.
4 Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403,
USA.
Author for correspondence (e-mail:
angie.ribera{at}uchsc.edu)
Accepted 2 August 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Motoneuron, Na+ channel, Axonal morphology, Zebrafish embryo
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Schneider and Granato, 2003).
Once targets are contacted, both activity-dependent and -independent processes
contribute to synapse formation, stabilization and maintenance (for a review,
see Sanes and Lichtman, 2001).
These findings have led to the traditional view that activity-independent
mechanisms guide axons prior to synapse formation.
Interestingly, outgrowing axons often form synapses at intermediate points
before they reach their final targets
(Lefebvre et al., 2004).
Further, spinal cord neurons exhibit spontaneous activity while their axons
are actively extending processes (for a review, see
Spitzer et al., 2004).
Moreover, disruption of normal patterns of spontaneous activity alters
neurotransmitter phenotypes, as well as motoneuron axonal pathfinding
(Borodinsky et al., 2004;
Hanson and Landmesser, 2004).
Thus, activity-dependent mechanisms influence pathfinding before axons reach
their final targets.
The role of activity is usually considered from the perspective of the
synapse, despite the fact that embryonic neurons are electrically excitable
prior to synapse formation (Spitzer et
al., 2004). In addition, intrinsic excitability of embryonic
neurons influences several key aspects of differentiation
(Gu and Spitzer, 1995;
Watt et al., 2000). However,
little is known about how motoneuron intrinsic excitability influences axon
outgrowth, especially while their processes navigate through the periphery
(but see Ming et al.,
2001).
A key determinant of neuronal excitability is the voltage-gated
Na+ channel. A spontaneously occurring mouse mutant, med
(motor endplate disease), displays a paralytic phenotype
(Duchen, 1970;
Duchen and Stefani, 1971). The
med mutation leads to inactivation of the Scn8a
Na+ channel 
-subunit gene that encodes the mammalian
Nav1.6 protein (Burgess et al.,
1995). The orthologous zebrafish gene, scn8a, encodes the
zebrafish Nav1.6a protein and is expressed early in the developing
spinal cord (Tsai et al.,
2001; Novak et al.,
2006a; Novak et al.,
2006b). Here, we test whether Nav1.6a regulates
development of embryonic spinal neurons. Nav1.6a knockdown alters
development of several, but not all, scn8a-expressing neurons.
Moreover, neuronal subtypes that do not express scn8a also display
developmental defects, revealing non cell-autonomous roles of ion channels
during neuronal development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Meng et al.,
1997; Gamse et al.,
2003). Twenty-two to 24 hours post fertilization (hpf) embryos
were manually dechorionated and transferred to embryo medium [EM; Westerfield
(Westerfield, 1995)]
containing 0.002% 1-phenyl-2-thiourea, to inhibit pigment formation.
Embryos were staged on the basis of external morphological criteria and age
is indicated by standard convention in terms of hours or days post
fertilization (hpf or dpf) (Kimmel et al.,
1995). Developmental landmarks that aided staging included somite
number, head-trunk angle, yolk sac size and position, and pectoral fin
buds.
Morpholino injections
Morpholino antisense oligonucleotides (MOs) were synthesized by Gene Tools
(Philomath, Oregon). The Nav1.6a MO (1.6MO) targeted the predicted
translation start methionine of Nav1.6a and had 25 residues with
the following sequence: 5'GGGTGCAGCCATGTTTTCATCCTGC-3'. Two other
1.6MOs, with sequences either slightly downstream or upstream, were also
synthesized. A fourth MO that targeted the splice junction between exons 3 and
4 was also synthesized. Similar results were obtained with the four different
(three translation-blocking and one splice-blocking) 1.6MOs, and the results
were pooled. 1.6MO-injected embryos are referred to as morphants. Two control
MOs (ConMOs), differing from a translation blocking or splice blocking MO by
five base mismatches, were synthesized. ConMO-injected embryos served as
controls. MOs were injected into the yolk at one- to two-cell at
concentrations ranging between 2.5 and 4 ng/nl in 1% Fast Green or
rhodamine-conjugated dextran.
Reverse transcription-polymerase chain reaction (RT-PCR)
RNA was collected from whole 24, 48 or 72 hpf embryos that had been
injected with either the splice blocking MO or its control MO using the TRIzol
Reagent (Invitrogen, Carlsbad, CA). cDNA synthesis was performed using oligo
dT (GIBCO-BRL, Gaithersburg, MD) and Superscript II reverse transcriptase (RT;
GIBCO-BRL). The PCR conditions were 5 minutes at 95°C; five cycles at
95°C for 1 minute, 50°C for 30 seconds, 72°C for 30 seconds; five
cycles at 95°C for 1 minute, 51°C for 30 seconds, 72°C for 30
seconds; 25 cycles at 95°C for 1 minute, 52°C for 30 seconds, 72°C
for 30 seconds; followed by 72°C for 7 minutes. The negative control
tested for amplification of genomic DNA and consisted of preparing an RT
reaction tube but omitting the RT enzyme. PCR products were gel purified and
cloned (PCR-Script Cloning Kit, Stratagene, La Jolla, CA). DNA sequencing
confirmed their identity.
In situ hybridization
Procedures were as described in Novak and Ribera
(Novak and Ribera, 2003). For
double in situ hybridization, the second probe was synthesized in the presence
of fluorescein-labeled nucleotide precursors (Roche); the probe was detected
with an anti-fluorescein antibody coupled to horseradish peroxidase, allowing
formation of a green fluorescent reaction product using tyramide based signal
amplification (Perkin Elmer, Boston, MA).
Immunocytochemistry
Procedures were as described previously
(Svoboda et al., 2001). We
used the primary antibodies listed in Table
1. The anti-Islet 39.4D5, 3A10, SV-2, zn-8, zn-12 and znp-1 mouse
monoclonals were developed by Drs T. Jessell (39.4D5. 3A10), J. Dodd (3A10),
K. Buckley (SV-2) and B. Trevarrow (zn-8, zn-12, znp-1), and were obtained
from the Developmental Studies Hybridoma Bank developed under the auspices of
the NICHD and maintained by The University of Iowa, Department of Biological
Sciences, Iowa City, IA 52242. Secondary antibody was applied during a
subsequent overnight incubation at 4°C (1:1000; anti-rabbit or anti-mouse
conjugated to either Alexa 548 or Alexa 488; Molecular Probes). Embryos were
squash-mounted on glass slides in 20 µl Prolong AntiFade Reagent (Molecular
Probes).
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) with the
following minor modifications. Embryos were injected at 19-21 hpf and fixed
1-3 hours later. Whole-mount preparations were incubated with rabbit
anti-fluorescein antibodies (Molecular Probes) and the SV-2 monoclonal
antibody (Table 1). Secondary
antibody application and subsequent processing was as described above.
Mosaic embryos
Mosaic embryos were created by injection of single cells in 2 to 3 hpf
wild-type of Tg(gata2:GFP) embryos with solution containing 0.3 mM MO and
lineage tracer (1-3% lysinated-rhodamine- or fluorescein-labeled dextran;
10,000 Mr). Embryos were fixed at 72 hpf and processed for
dextran and GFP or zn-8 immunoreactivities (see below).
Visualization of RNA in situ hybridization signals or antibody labeling
Embryos were viewed on either a Nikon Eclipse TE2000 inverted scope at
40x or a Zeiss Pascal Confocal microscope at 10, 40 or 63x. Images
were acquired digitally and processed using Adobe Photoshop Software (Mountain
View, CA). For confocal imaging, the pinhole was set to one Airy unit.
Rhodamine/Alexa-568 and Fluorescein/Alexa 488 were visualized using separate
channels. Z-stacks were collected at 1-1.5 µm intervals; data are
presented as single sections unless indicated otherwise. Because embryos were
squash-mounted, it was possible to view motor nerves from both sides of an
embryo in a single section (e.g. Figs
5,
7). To determine the percent of
pixels in a field that showed signals above threshold for both channels, we
used the colocalization algorithm of the Zeiss Pascal LSM software.
Electrophysiology
Embryos dissections and nucleated patch recordings were performed as
described previously (Ribera and
Nüsslein-Volhard, 1998;
Pineda et al., 2005). We used
nucleated patches to avoid space clamp problems. The recording solution
consisted of: 127 mM NaCl, 20 mM TEA-Cl, 3 mM KCl, 10 mM CoCl2, and
10 mM HEPES (pH 7.2). Electrodes had resistances of 2-3 M
when filled
with pipet solution [125 mM CsCl, 12 mM NaCl, 10 mM EGTA and 10 mM HEPES (pH
7.2)]. Recordings were made using an Axopatch 200B Amplifier (Axon
Instruments) and were continued when the following criteria were met online:
(1) input resistances greater than 1G; and (2) mono-exponential decay
of the whole cell capacitance transient. The pCLAMP9 and Origin programs (Axon
Instruments) were used to analyze data.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Henion et al., 1996).
Ventral spinal neurons also contained scn8a transcripts
(Fig. 1D-G). The position of
ventral scn8a+ cells suggested that some might be
motoneurons. Primary motoneurons (PMNs) are born during gastrulation, whereas
secondary motoneurons (SMNs) arise from later rounds of neurogenesis
(Beattie et al., 1997). To
identify the PMN subtype(s) that expressed scn8a, we used specific
markers of PMN subtypes and the Tg(flh:GFP) transgenic line
(Gamse et al., 2003). By 24
hpf, PMN subtypes can be distinguished on the basis of their axonal
trajectories (for a review, see Lewis and
Eisen, 2003): RoP axons innervate the region around the horizontal
myoseptum. MiPs have axons that project dorsally, whereas CaP axons innervate
ventral trunk muscle. Tg(flh:GFP) transgenic embryos displayed GFP in CaPs
(arrowheads), identified on the basis of ventrally projecting axons (arrows)
(Fig. 1D)
(Gamse et al., 2003). The
Islet (Isl) antibody recognizes Islet1 as well as Islet2 transcription factors
found in RB cells (asterisks) and PMNs (arrowheads)
(Fig. 1D). However, MiPs and
CaPs differentially express islet transcription factor genes, and
isl1 mRNA localizes to MiPs, whereas isl2 transcripts are
present in CaPs (Appel et al.,
1995; Inoue et al.,
1994; Tokumoto et al.,
1995). Consistent with this, GFP- neurons in the
Tg(flh:GFP) line expressed isl1 mRNA, a marker of MiPs
(Fig. 1E, carats). Thus, CaPs
are the GFP+ PMNs in Tg(flh:GFP) embryos. Furthermore, in situ
hybridization revealed that scn8a transcripts localized to the
GFP+ CaPs (Fig. 1F).
Consistent with this, double in situ hybridization studies indicated that
CaPs, identified on the basis of isl2 mRNA
(Fig. 1G, green), also
expressed scn8a (Fig.
1G, red). Thus, CaPs are the PMN subset that expressed
scn8a.
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). scn8a mRNA was detected in GFP+ SMNs of
at both 36 and 72 hpf (Fig.
2A,C). Interestingly, despite expression of scn8a in
CaPs, ventrally projecting SMNs did not express the gene. In Tg(gata2:GFP)
embryos, GFP identifies SMNs with axons that project ventrally, similar to
CaPs (Meng et al., 1997).
However, these GFP+ SMNs did not display scn8a at either
36 or 72 hpf (Fig. 2B,D). In
sum, only a subset of PMNs and SMNs expressed scn8a. Moreover, the
PMN and SMN populations that expressed scn8a differed with respect to
axonal trajectories.
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). The
efficacy of the splice blocking MO was tested by RT-PCR. The splice blocking
MO targeted the junction between the third and fourth exons. In RNA extracted
from 24, 48 and 72 hpf morphant embryos, the 96 bp intron between these exons
was retained until at least 72 hpf (Fig.
3A). Direct sequencing of the PCR product confirmed that the 96 bp
intron had been retained, resulting in a premature STOP codon (results not
shown). By contrast, in control-injected embryos, normal splicing
occurred.
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In addition to examining protein expression, we assessed the effects of the
1.6 and ConMOs by recording voltage-gated Na+ current. Injection of
the ConMO had no effect on voltage-gated Na+ current
(INa) recorded from RB cells of 48 hpf control embryos
(Fig. 3F). By contrast, RB
cells in 48 hpf morphants displayed either no INa or one of
dramatically reduced amplitude (Fig.
3F,G). On average, peak INa amplitude was 25% that of
control (Fig. 3G). The
electrophysiological data were consistent with the previously described
touch-insensitive behavioral phenotype of 1.6MO morphants
(Pineda et al., 2005). We also
found that 1.6MO morphants became increasingly more immotile with time and
displayed little spontaneous swimming after 72 hpf.
In summary, two different measures of Na+ channel protein, immunoreactivity and current, were reduced in scn8a expressing neurons following injection of 1.6, but not control, MO. These results indicated that 1.6MO injection was an effective strategy for Nav1.6a knockdown in the developing embryo.
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). As a
result of the diminished Na+ current amplitudes in RB cells of
morphants (Fig. 3F,G), we
predicted that RB cells would survive longer in morphant versus control
embryos. At 72 hpf, examination of RB cell bodies revealed that injection of
1.6, but not control, MO led to an increase in RB survival
(Fig. 4), as expected because
of the loss of Na+ current (Fig.
4) (Svoboda et al.,
2001).
Axonal trajectories of motor neurons were abnormal in 1.6MO morphants embryos
In addition to RB cells, specific populations of motoneurons expressed
scn8a. CaPs and dorsally projecting SMNs expressed the gene (Figs
1,
2). Recent studies provided
evidence that perturbation of activity in all embryonic spinal neurons during
stages of neurite outgrowth altered neurotransmitter expression
(Borodinsky et al., 2004).
However, the extent to which activity regulates the development of motoneurons
is poorly understood. The restricted pattern of scn8a expression in
select motoneuron populations allowed us to examine the role of activity in
development of specific zebrafish motoneurons.
We examined morphological development of motoneurons using znp-1 and SV-2,
antibodies that recognize proteins present in both PMNs and SMNs
(Fig. 5). In addition, we
assessed target recognition by comparing the location of antibody labeling to
that of postsynaptic receptors detected by -bungarotoxin. At 48 hpf,
little difference was noted between ventrally projecting axons of 1.6MO
morphants versus control injected embryos
(Fig. 5A,B). However, by 72
hpf, morphologies of motoneuron axons differed in 1.6MO morphants versus
controls (Fig. 5C,D). In
morphant embryos, axons branched more. Moreover, in controls, the most
distally labeled region of motoneuron axons typically were aligned with
postsynaptic receptors by 72 hpf (Fig.
5C) (LeFebvre et al.,
2004; Panzer et al.,
2005). By contrast, in morphants, the distal processes of several
motoneuron axons did not align with postsynaptic receptors
(Fig. 5D). Analysis of the SV-2
and -bungarotoxin signals in the distal regions of axons of control and
morphant embryos indicated 84 versus 71% colocalization at 72 hpf
(n=9 and 11, respectively; P<0.0004). Thus, the results
indicated that 1.6MO injection altered morphological development of motoneuron
axons and their contacts with muscle cells.
After 
28 hpf, both PMN and SMN axons contribute to the ventral
projections but SV-2 and znp-1 antibodies recognize both types of axons
(Pike et al., 1992;
Ott et al., 2001). Thus, SMN,
PMN, or both types of axons, could be defective. Because CaPs but not
ventrally projecting SMNs expressed scn8a (Figs
1,
2), we expected that the
defects in motoneuron axonal trajectories detected by SV-2 or znp-1 were
consequent to effects of 1.6MO on CaP axons. Furthermore, because PMN axons
serve as pioneers and establish a scaffold for SMN outgrowth (for a review,
see Lewis and Eisen, 2003),
abnormal CaP axons would induce aberrant morphology of SMN axons
(Pike et al., 1992).
To test whether CaP axons were affected by injection of the 1.6 MO, we
examined their ventral projections. At 21-24 hpf, the patterns of znp-1
immunoreactivity did not differ between 1.6 and control morphant embryos
(Fig. 5E-H). Furthermore,
individually dye-labeled CaP axons did not differ between 1.6 and control
morphant embryos (Fig. 5I,J).
Moreover, morphological aspects of synapse formation also appeared to be
similar on the basis of the relative positions of SV-2 and
-bungarotoxin signals (Fig.
5K,L). Thus, no defects were noted in CaP axon morphology as a
result of the 1.6MO injection, implicating SMN axons in the defective ventral
projections present in 1.6MO morphants.
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; Ott et al.,
2001). Beginning at 30 hpf, neurolin is initially detected in
both the cell bodies and emerging axons of SMNs
(Fig. 6A). By 72 hpf, cell body
expression is downregulated but axonal labeling persists
(Fig. 6E)
(Ott et al., 2001). Typically,
at the choice point near the horizontal myoseptum, the motor nerve forms three
distinct branches by 66 hpf (Fig.
6C) (Fashena and Westerfield,
1999; Ott et al.,
2001). One branch contains axons of dorsally projecting SMNs.
(Fig. 6C, arrowheads). The
second branch contains axons of ventrally projecting SMNs
(Fig. 6C, asterisks). A third
branch projects rostrally within the vicinity of the horizontal myoseptum
(Fig. 6C, arrows).
At 48 hpf, little difference was observed in zn-8 immunoreactivity between
1.6MO- and control-injected embryos (Fig.
6A versus B). However, at this stage only the common branch of all
axons to the myoseptum choice point was present. At 66 hpf
(Fig. 6C,D), 1.6MO morphants
consistently lacked the rostrally projecting nerves that were now present in
controls (arrows). In addition, SMN axons that projected dorsally were not
detected by zn-8 at 66 hpf in 1.6MO morphants although they were present in
all segments of controls (Fig.
6C versus D; arrowheads). At 78 hpf, rostral branches were still
absent in some segments in 1.6MO morphants
(Fig. 6F, arrows). In addition,
some dorsal segments still lacked dorsally projecting SMN axons
(Fig. 6F, arrowhead). We
followed recovery of innervation of the dorsal musculature to later times
(Fig. 6G). By 144 hpf, recovery
of dorsal muscle innervation was still not complete. The time course of
recovery is consistent with that expected for turnover of MOs
(Nasevicius and Ekker, 2000)
(see Discussion).
As recovery of rostral and dorsal SMN motor nerves occurred, the axons of ventrally projecting SMNs began to display abnormal properties (Table 2). In 88% (21/24) of 1.6MO morphants, more than one ventrally projecting nerve was present in at least one segment of the embryo (Fig. 6H). Such additional ventral roots were not observed in control embryos. In addition, the distal ends of ventrally projecting SMN axons branched more and appeared more splayed than did those in control injected embryos (Fig. 6I,J, asterisks; Table 2). This phenotype was not observed in control injected embryos (0/32) but was present in 54% (13/24) of 1.6MO morphant embryos. Interestingly, in the three 1.6MO morphants embryos that did not display an additional ventral root, the branching phenotype was present. Thus, all 1.6MO morphants displayed at least one of two different ventral phenotypes.
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).
Mauthner cell axons project caudally in the spinal cord in the vicinity of
motoneuron cell bodies and initial axon segments
(Kimmel et al., 1982) but do
not express scn8a (Novak et al.,
2006a). Mauthner neurons of 1.6MO morphants appeared normal
(Fig. 6K-N). Their somata were
normal in shape and size, and their contralaterally projecting axons displayed
normal trajectories. At 72 hpf, a time when defects in SMN axons were detected
in 1.6MO morphants, Mauthner cells axons had normal trajectories within the
spinal cord (Fig. 6M,N). These
results indicated that the 1.6MO did not have a non-specific general effect on
axonal growth in the spinal cord. Another possibility that we considered is that 1.6MO injection altered expression of neurolin, as detected by zn-8 labeling, but did not perturb axonal outgrowth directly. Typically, neurolin is downregulated in SMN cell bodies by 72 hpf. However, in 1.6MO morphants, neurolin persisted in SMN cell bodies at 72 hpf, supporting this possibility (Fig. 6F). To avoid this potential problem, we examined axonal trajectories in the Tg(gata2:GFP) line (Fig. 7). In Tg(gata2:GFP)-treated embryos, GFP+ ventrally projecting SMN axons (Fig. 7B,D,E) displayed phenotypes similar to those detected by zn-8 immunocytochemistry (Fig. 6). Specifically, GFP+ axons branched much more extensively in morphant embryos (Fig. 7B,D,E) than they did in controls (Fig. 7A,C). These results indicate that, despite the lack of detection of scn8a in ventrally projecting SMNs, their axonal trajectories were perturbed by injection of 1.6MO.
Mosaic analysis reveals cell non-autonomous effect of 1.6MO on ventrally projecting SMNs
The results presented above suggest that Nav1.6a channels exert
non-autonomous effects on axons of ventrally projecting SMNs. If so, ventrally
projecting SMNs would develop normally if they did contain the 1.6MO but other
cells did not. Conversely, ventrally projecting SMNs would show defects if
they did not contain the 1.6MO but other cells did. To test directly for
non-cell autonomous effects of Nav1.6a, we performed mosaic
analysis by injecting single cells of 2-3 hpf embryos with 1.6MO and lineage
tracer (Fig. 8).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Voltage-gated Na+ channels reside in the plasma membrane and
provide an entry point for extracellular Na+ to the neuronal
cytoplasm. Their functional elimination would be expected to result in
cell-autonomous effects. Consistent with this, RB Na+ current
amplitude and cell death were reduced (Figs
3,
4), and dorsally projecting
SMNs displayed a significant delay in axon outgrowth in morphant embryos
(Fig. 6). The latter phenotype
recovered with a time course expected for MO turnover
(Nasevicius and Ekker, 2000),
suggesting that blockade of Nav1.6a translation prior to 72 hpf
prevented axon outgrowth from these SMNs; later, when MO concentrations fell,
translation of Nav1.6a began, allowing recovery of axon
outgrowth.
Some neurons that express scn8a did not display a phenotype.
Notably, CaPs expressed scn8a but developed normal axonal
trajectories when Nav1.6a was knocked down
(Fig. 5). Interestingly, CaP
axons also developed normally in nic1b107 mutants, that
are immotile as a result of a null-type mutation in the -subunit of the
postsynaptic muscle acetylcholine receptor
(Liu and Westerfield, 1990;
Westerfield et al., 1990;
Sepich et al., 1998). By
contrast, a different mutation in the muscle acetylcholine receptor
-subunit gene (twister; nic1twister
dbn12), results in excessive activity and twister mutants
move in an uncoordinated manner (Lefebvre
et al., 2004). Furthermore, unlike nic1b107
mutants, twister mutants display abnormal PMN axonal trajectories and
muscle degeneration (Lefebvre et al.,
2004). Thus, blockade of the muscle postsynaptic receptor has no
effect on CaP axons, whereas hyperactivity results in defective CaP axons.
Surprisingly, Nav1.6a knockdown led to abnormal axon outgrowth in an unexpected motoneuron subtype, the ventrally projecting SMN, that did not display scn8a expression (Fig. 2). Both immunocytochemical staining (zn-8) as well as direct GFP labeling [Tg(gata2:GFP)] revealed abnormal axonal morphologies, eliminating the possibility that the phenotype resulted from abnormal expression of neurolin (Figs 6, 7). CaP axons were also normal (Fig. 5), ruling out abnormal pioneer axon morphology as a basis for the defective SMN axonal trajectories. These results implicated Na+ channels unexpectedly in non-cell-autonomous as well as cell-autonomous developmental roles, a conclusion that was further supported by mosaic analyses.
With respect to the mosaic analyses, one potential caveat is that although morpholino knockdown significantly reduces Nav1.6a protein levels, it might not entirely eliminate it. If this were the case and if reduced levels of Nav1.6a sufficed to achieve its function in ventrally projecting SMNs, our mosaic experiments might incorrectly suggest that Nav1.6a did not cell-autonomously affect this cell type (e.g. Fig. 8B). However, the mRNA encoding Nav1.6a, scn8a, was not detected in ventrally projecting SMNs even though it was present in dorsally projecting SMNs (Fig. 2), suggesting that 1.6MOs had no effect on ventrally projecting SMNs because these neurons did not express the targeted gene. Moreover, morpholino knockdown of Nav1.6a protein in cells other than ventrally projecting SMNs sufficed to reveal non cell-autonomous effects (Fig. 8C,D).
Previous studies revealed that absence of PMNs had complex effects on SMNs
that differed depending on whether SMNs projected axons dorsally or ventrally
(Pike et al., 1992). The
results suggested that the axons of different SMN subtypes have distinct
abilities to recognize guidance cues (Pike
et al., 1992). Our present results are consistent with this idea.
Pike et al. (Pike et al.,
1992) proposed that cues on PMN axons are directly responsible for
some aspects of SMN axon guidance. In light of recent studies demonstrating
the importance of developmentally regulated modification of the substrata on
which motor axons navigate (Zhang et al.,
2004; Schneider and Granato,
2006), another possible interpretation is that in the absence of
Nav1.6a, even though CaP axons extend normally, they fail to
interact correctly with their environment; consequently, the environment is
altered so that SMN axons cannot extend properly, or interactions between the
CaP and SMN somata are altered, resulting in failure of the SMNs to extend
axons properly. These possibilities can be addressed in future
experiments.
Even though morpholinos have been used with great success in zebrafish, not
all morpholinos work as expected, requiring control experiments such as rescue
by RNA overexpression, reduction of targeted protein levels or function, use
of appropriate control morpholinos and/or RT-PCR analysis of mRNA when using
splice-blocking morpholinos (Nasevicius
and Ekker, 2000). Rescue experiments typically involve injection
of the targeted RNA immediately after injection of the MO. However,
scn8a RNA overexpression by itself produced phenotypes similar to
those produced by 1.6MO injection (e.g. reduced touch sensitivity). In these
experiments, the injected scn8a RNA is expected to be present in
cells that normally express the gene, as well as in cells that do not, and
ectopic expression of scn8a might have resulted in abnormal function
of the relevant circuits. Moreover, rescue by scn8a injection was
complicated by the fact that injection of both 1.6MO and scn8a mRNA
resulted in profound embryonic lethality, with few surviving severely deformed
embryos. These effects of scn8a overexpression either alone or in
combination with 1.6MO prevented interpretation of scn8a rescue
experiments.
Several other standard control experiments were successful. For each 1.6MO,
we compared results to those obtained with a specific control morpholino that
differed from a 1.6MO by four or five base mismatches (see Materials and
methods). We used three different translation-blocking MOs that had
dose-dependent effects (Pineda et al.,
2005). In addition to the three different translation blocking
morpholinos, we also used a splice-blocking morpholino along with its own
specific control morpholino. The splice-blocking morpholino, but not its
control, resulted in incorrect processing of scn8a mRNA, even at 72
hpf (Fig. 3A). 1.6MO injection
resulted in reduced Na+ channel levels, detected either
immunocytochemically (Fig.
3B-E) or electrophysiologically
(Fig. 3F,G). Moreover, 1.6MOs
mimicked a previously reported effect of reduced Na+ channel
function - reduction of RB cell apoptosis
(Fig. 4)
(Svoboda et al., 2001). In
summary, several lines of evidence support the conclusion that the 1.6MO
effects resulted from Nav1.6a knockdown.
The small number and earlier development of PMNs versus SMNs
(Beattie et al., 1997) has
facilitated studies of PMN axon guidance mechanisms. Despite the limited data
regarding SMNs, it is clear that PMNs and SMNs share some aspects of axon
guidance mechanisms because both rely upon myotomal derived signals
(Zeller et al., 2002). In
addition, a few studies have provided evidence for SMN-specific axon
growth/guidance mechanisms. For example, neurolin is required for normal
development of SMN but not PMN axons (Ott
et al., 2001). Further, nicotine exposure affects morphology of
SMN axons (Svoboda et al.,
2002). However, nicotine exposure began at 22 hpf, well after
initiation of, and possibly too late for, effects on PMN axon outgrowth. A
recently isolated mutant, where's waldo (wdo), displays
defects in SMN axon outgrowth, and many segments lack the SMN component of the
dorsal nerve similar to 1.6MO morphants
(Panzer et al., 2005) (this
study). In light of our findings, the effects of muscle inactivity
(nic1b107) and hyperactivity (twister) on SMN
axon morphology both warrant examination.
The mammalian gene Scn8a is orthologous to scn8a
(Novak et al., 2006b). The
mouse mutant med lacks a functional Scn8a gene and displays
aberrant sprouting of motor nerve terminals similar to the highly branched
axons of ventrally projecting SMNs of 1.6MO morphants (Figs
5,
7)
(Duchen, 1970;
Burgess et al., 1995).
med mutants also show neonatal hindlimb paralysis
(Duchen, 1970). Similarly,
1.6MO morphants became severely immotile at 5 dpf, suggesting a progressive or
delayed deterioration of the neuromuscular system. Although the defects that
we report here would be expected to reduce motility of 1.6MO morphants, it is
not clear why 1.6MO morphants developed such extensive paralysis, especially
at stages after extensive MO turnover. If there were early crucial periods for
the developmental roles of Nav1.6a, not all phenotypes would
recover, perhaps leading to degeneration of neuromuscular junctions. In
addition, larvae use PMNs primarily for escape responses and SMNs primarily
for swimming (Liu and Westerfield,
1990). Because ventrally projecting SMNs are affected in 1.6MO
morphants, the entire ventral musculature may not contract sufficiently to
allow normal swimming. Further studies of 3 dpf or older larvae are needed to
resolve this issue.
In summary, our data indicate that knockdown of a specific Na+
channel isotype affects SMN axon outgrowth. The effects of Nav1.6a
knockdown on SMN axon outgrowth occur in cells that express scn8a
(e.g. dorsally projecting SMNs) as well as those that do not (e.g. ventrally
projecting SMNs), indicating both cell autonomous and non-autonomous effects
of a voltage-gated Na+ channel on axonal development. It is not yet
known how Nav1.6a-dependent effects are spread to neurons that do
not express the scn8a gene. The underlying mechanisms might involve
altered secretion of neuronal neurotrophic or muscle retrograde factors (see
above) and/or the extensive electrical coupling that is present in the
embryonic spinal cord (Saint-Amant and
Drapeau, 2000; Saint-Amant and
Drapeau, 2001). Overall, the results demonstrate that
voltage-gated neuronal Na+ channels play developmental and
conventional excitability roles in the vertebrate nervous system.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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0.001).
