1 Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute,
2-1, Hirosawa, Wako, Saitama 351-0198, Japan
2 Center for Molecular and Cellular Biology, The University of Queensland,
Queensland 4072, Australia
3 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation (JST), 3-4-5 Nihonbashi, Chuo-ku, Tokyo 103-0027,
Japan
4 Graduate School of Biological Sciences, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan
Author for correspondence (e-mail:
hitoshi{at}brain.riken.go.jp)
Accepted 2 April 2004
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SUMMARY |
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Key words: Zebrafish, PlexinA4, Slit, Islet2, LIM/homeodomain protein, Axon branching, Zebrafish
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Introduction |
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As Islet2 is a transcription factor, downstream target genes of Islet2 must
be involved in this particular process. To determine the molecular mechanisms
for the asymmetric development (peripheral versus central axons) of the
primary sensory neurons and its control by Islet2 in zebrafish embryos, we
focused on one of the cDNA fragments (D204) that was originally identified as
a consequence of ordered differential display (ODD) screening, in order to
identify the genes specifically downregulated in the tissue around the
midbrain-hindbrain boundary (MHB) by overexpression of LIMIsl-3 in
the embryo (Hirate et al.,
2001). Besides expression in the midbrain, D204 is normally
expressed in Rohon-Beard neurons and the trigeminal sensory ganglion neurons,
and its expression in these primary sensory neurons is downregulated by
overexpression of either LIMIsl-3 or LIMIsl-2
(Fig. 1A,A',C,C'),
suggesting that the gene encoded by D204 may act as a downstream target of
Islet2.
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Materials and methods |
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Isolation RNA and cDNA library construction
Total RNA from 22-26 hours post fertilization (22-26 hpf) zebrafish embryos
was isolated by using ULTRASPEC RNA isolation kit (BIOTECX Laboratories) and
poly(A)+RNA was selected using oligo(dT) DYNAbeads (Dynal Biotech).
Double-stranded cDNA from 5 µg of poly(A)+RNA was synthesized
with oligo(dT) and random hexamer primers mixture and cloned into the
gt11 vector using SuperscriptII choice system (Gibco BRL, Life
Technologies). We obtained 6x106 independent
phage
clones.
cDNA cloning and sequencing
We screened 22-26 hpf zebrafish embryo cDNA libraries as described in
Sambrook and Russell (Sambrook and
Russell, 2001) using 582 bp PCR fragment of D204 that was
identified by ODD comparing wild-type and LIMIsl-3-overexpressing
embryos (Hirate et al., 2001
).
Seventeen independent positive clones were isolated from 106
recombinant clones. Restriction fragments of cDNA were cloned into
pBluescriptII (Stratagene), and sequenced by using Thermo Sequenase cycle
sequencing kit (Amersham Pharmacia Biotech) and BigDye terminator cycle
sequencing kit (Applied Biosystems). Sequences were determined by GENETYX-MAC
9.01 program (Software Development). The Accession Number for the zebrafish
plexinA4 cDNA is AB103158. Homology searches were performed using
BLAST algorithm at the NCBI (Altschul et
al., 1990
). The phylogenic tree was constructed using CLUSTAL W
multiple sequence alignment program at the GenomeNet
(http://clustalw.genome.ad.jp/).
Mouse (Kameyama et al., 1996a
;
Kameyama et al., 1996b
;
Suto et al., 2003
) and
Xenopus (Ohta et al.,
1995
) PlexinA family were used for comparison.
Whole-mount in situ hybridization and immunohistochemistry
Whole-mount in situ hybridization was performed according to the standard
method (Westerfield, 2000).
The 1560 bp fragment of plexinA4 cDNA that includes 580 bp of
3' untranslated region (UTR) was used as a template for synthesizing the
antisense RNA probe. Antisense RNA probe was synthesized using the
digoxigenin-RNA labeling kit (Roche Diagnostics). The immunohistochemistry and
double staining were performed as described previously
(Segawa et al., 2001
;
Yeo et al., 2001
). The
dilution rate of primary antibodies was 1:2000 for the anti-acetylated
-tubulin antibody (Sigma), 1:500 for anti-GFP antibody (Santa Cruz
Biotechnology) and 1:500 for 3A10 monoclonal antibody (Developmental Studies
Hybridoma Bank at the University of Iowa).
Plasmids construction
The plasmids for generating transgenic zebrafish of dnPlexinA4
(pSS-hsp70:dnPlexinA4-GFP) or PlexinA4 (pSS-hsp70:PlexinA4-GFP) were
constructed in the following manner. The backbone plasmid was pBluescript II
SK. The NheI-SspI fragment of pIRES2-EGFP vector (Clontech)
was inserted between the XbaI and filled-in NotI sites of
pBluescript II SK to create pSK-IRES2-EGFP. The EcoRI-PstI
fragment of the zebrafish heat-shock protein 70 (hsp70)
promoter (Halloran et al.,
2000) was inserted between the EcoRI and PstI
sites of pSK-IRES2-EGFP to create pHsp-IRES2-EGFP. Then, the
XhoI-EcoRI fragment of the primary sensory neuron-specific
enhancer (SS) of the zebrafish islet1 gene
(Higashijima et al., 2000
) was
inserted between the XhoI and EcoRI sites of
pHsp70:IRES2-EGFP to create pSS-hsp70:IRES2-EGFP.
The 2280 bp BamHI-XbaI fragment that contains the
extracellular region of plexinA4 was inserted between the
BamHI and XbaI sites of pCS2+ vector
(Turner and Weintraub, 1994).
The 1604 bp BamHI fragment that contains short 5'UTR, the first
ATG and the extracellular region of plexinA4 were inserted to the
BamHI site of this plasmid to create pCS2+dnPlexinA4. The
HindIII site of the HindIII-SnaBI fragment of
pCS2+dnPlexinA4 was filled-in by Klenow fragment of DNA polymerase, and this
fragment was inserted into the SmaI site of pSS-hsp70:IRES2-EGFP to
create pSS-hsp70:dnPlexinA4-IRES2-EGFP.
To construct recombinant plasmids encoding the chimeric proteins of PlexinA4 and dnPlexinA4 fused with green fluorescent proteins (GFP), polymerase chain reaction (PCR) amplifications of the 3' region of PlexinA4 and dnPlexinA4 were performed using plexinA4 cDNA as a template, respectively, with the following sets of oligonucleotide primers; PlexA4-5', 5'-TAT ACT ACC CAA ATC CTG TG-3'; and dnPlexA4-3', 5'-CTT GCC CAT GGA GGC AAT TAA GA-3' or PlexinA4-3', 5'-AAA TGT GGC CAT GGG CTC TC-3' (underlines indicate the NcoI sites for ligation with GFP cDNA fragment at its first ATG). The amplified DNA fragment was subcloned into pGEM-T Easy vector (Promega) to generate pGEM-3'dnPlex and pGEM-3'Plex. The SacI-SacII fragment of pSS-hsp70:dnPlexinA4-IRES2-EGFP was inserted between the SacI and SacII sites of pBluescriptII KS, and the SacI-NcoI fragment of pGEM-3'dnPlex was subcloned between the SacI and NcoI sites of this plasmid. This plasmid contains the C-terminal region of PlexinA4 fused with GFP in the PmaCI-NotI fragment. This PmaCI-NotI fragment was subcloned between the PmaCI and NotI sites of pSS-hsp70:dnPlexinA4-IRES2-EGFP to create pSS-hsp70:dnPlexinA4-GFP. The XbaI-NcoI fragment of pGEM-3'Plex was inserted between the XbaI and NcoI sites of pSS-hsp70:dnPlexinA4-GFP to create pSS-hsp70:PlexinA4-GFP.
The plasmid for single cell imaging (pSSICP-Kaede) was constructed by
replacing the GFP cDNA of SSICP-GFP
(Higashijima et al., 2000)
with Kaede cDNA (kindly provided by Dr A. Miyawaki, Brain Science Institute,
Riken, Japan) (Ando et al.,
2002
).
Generation of the transgenic zebrafish
Constructed plasmids were purified using QIAprep Spin Miniprep kit
(QIAGEN). DNA solution of 20 µg/ml in distilled water was microinjected
into one- to four-cell stage zebrafish embryos. The injected embryos were
raised to sexual maturity, and crossed with each other to identify the founder
fish pairs that bear the progeny transgenic for pSS-hsp70:dnPlexinA4-GFP.
Transgenic embryos were identified by their expression of the GFP fluorescence
in the trigeminal sensory ganglion neurons and Rohon-Beard neurons that was
induced by sensory neuron-specific enhancer of the zebrafish Islet1
and in the whole body following the heat shock treatment at 39°C for 45
minutes. Two lines, termed
Tg(SS-hsp70:dnPlexinA4-GFP)rw016a and
Tg(SS-hsp70:dnPlexinA4-GFP)rw016b, in which dnPlexinA4-GFP
fusion protein was induced, were used for further analysis.
Injection of morpholino oligonucleotide
Specific 25-base antisense morpholino oligonucleotide (AMO) (Gene Tools)
against plexinA4 was designed to contain the first start codon (ATG),
and control morpholino oligonucleotide (MO) with four-base mismatch was
designed. Their sequences were as follows: AMO against plexinA4 mRNA,
5'-TCCAT CTCCT ATTGT GAAAA GCCAT-3'; control MO, 5'-TCCtT
CTCgT ATTGT GAAtA GCgAT-3' (where the mismatch bases are indicated by
lowercase letters). They were dissolved in Danieou buffer as instructed by
Nasevicius and Ekker (Nasevicius and
Ekker, 2000) at the concentration of 2 mg/ml and microinjected by
air pressure into the one- to four-cell stage zebrafish embryos.
Heat shock treatment of transgenic zebrafish
Heterozygous embryos of transgenic line
Tg(hsp70:Slit2-GFP)rw015d (previously called HS2E-4S)
(Yeo et al., 2001) were used
for Slit2-GFP-overexpression experiments. Double transgenic zebrafish embryos,
which can overexpress both Slit2-GFP and dnPlexinA4-GFP, were obtained from
crossing Tg(hsp70:Slit2-GFP)rw015d homozygous with the
Tg(SS-hsp70:dnPlexinA4-GFP)rw016b homozygous fish. These
embryos were maintained at 39°C for 45 minutes to induce the transgene
expression at 14 hpf.
Time-lapse observation of axonal outgrowth of a single primary sensory neuron expressing Kaede
A part of the embryonic spinal cord expressing Kaede in Rohon-Beard neurons
was exposed to UV light using the 40x water immersion objective lens
(ACHROPLAN 40X/0.80w, Carl Zeiss) and the filter set 01 (BP 365/12, FT 395, LP
397. Carl Zeiss) of Zeiss LSM510 until the emitted light turns from pale blue
to red. Confocal images were captured from 23 hpf and 27 hpf embryos using the
same microscope.
Scoring branching of the peripheral axons of Rohon-Beard neurons
Embryos were stained with anti-acetylated -tubulin antibody (Sigma).
Camera lucida drawings of the peripheral axons of Rohon-Beard neurons were
made. We divided the trunk region into six longitudinal areas along the
dorsoventral axis as shown in Fig.
4G, and counted the number of branching points of the peripheral
axons of Rohon-Beard neurons in each region caudal to the transition between
the yolk and yolk extension.
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Results |
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Molecular cloning of full-length D204 revealed that it encodes the zebrafish ortholog of PlexinA4
Sequence analysis of a full-length cDNA fragment for D204 and subsequent
BLAST homology search revealed that D204 encodes a member of PlexinA family.
Phylogenic tree analysis among mouse PlexinA family, Xenopus Plexin
and zebrafish PlexinA revealed that D204 encodes the zebrafish ortholog of
PlexinA4 (Fig. 2A)
(Suto et al., 2003). Proteins
of PlexinA family consist of a Sema domain, Met related sequence/Plexin,
semaphorin, integrin domain (MRS/PSI domain), glycine-proline
rich/immunoglobin-like fold shared by plexins and transcription factors motif
(G-P/IPT motif) and intracellular Plexin family conserved Sex Plexin domain
(SP domain) (Bork et al.,
1999
). The overall identity of zebrafish PlexinA4 in the amino
acid sequence to mouse PlexinA4 is 67.6%. The extracellular region shows an
average of 59.5% identity with Sema domain, MRS/PSI domain and G-P/IPT motif,
showing 60.4%, 61.5% and 55.9% identity, respectively. The intracellular
region motif shows an average of 83.5% identity with three conserved domains,
the domain 1-3, respectively, showing 85.5%, 83.3% and 81.8% identity
(Fig. 2B).
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We transiently expressed PlexinA4 fused with green fluorescent protein
(GFP) at the C terminus (PlexinA4-GFP), in both the trigeminal sensory
ganglion neurons and Rohon-Beard neurons by injecting a plasmid,
pSS-hsp70:PlexinA4-GFP (Fig.
3D), into the one-cell stage embryos, which drives expression of
PlexinA4-GFP fusion protein under control of the primary sensory
neuron-specific enhancer (SS) of the zebrafish islet1 gene at room
temperature (Higashijima et al.,
2000). The PlexinA4-GFP fusion protein is distributed both in the
central and peripheral axons of both the trigeminal sensory ganglion neurons
(Fig. 3E) and Rohon-Beard
neurons (Fig. 3F).
PlexinA4 protein lacking the cytoplasmic region is known to act as a
dominant-negative variant (Takahashi et
al., 1999; Rohm et al.,
2000
). A stable zebrafish line
Tg(SS-hsp70:dnPlexinA4-GFP) transgenic for pSS-hsp70:dnPlexinA4-GFP
driving expression of this dominant-negative variant fused with GFP
(dnPlexinA4-GFP) under control of the same primary sensory neuron-specific
enhancer was also created (Fig.
3G). In the embryos of this line, dnPlexinA4-GFP fusion protein
was expressed both in the trigeminal sensory ganglion neurons
(Fig. 3H) and Rohon-Beard
neurons (Fig. 3I), and they
were also distributed both in the central and peripheral axons.
PlexinA4 promotes branching in the peripheral axons of Rohon-Beard neurons
As the promoter of the zebrafish heat-shock protein 70
(hsp70) gene was used as a core promoter for creating these
transgenic fish, heat shock treatment can induce ubiquitous overexpression of
dnPlexinA4-GFP in addition to the basal expression specifically observed in
the primary sensory neurons (Halloran et
al., 2000) (Fig.
3J). Specific AMO (Nasevicius
and Ekker, 2000
) was designed for plexinA4 mRNA. We
checked its activity by examining whether it can prevent the translation of
dnPlexinA4-GFP fusion protein after heat shock treatment in this transgenic
embryo when AMO was injected at the one-cell stage. In transgenic embryos,
heat-induced dnPlexinA4-GFP signals were detected, and signals of in situ
hybridization for gfp were also detected ubiquitously. However, in
AMO-injected transgenic embryos, no GFP signals were detected after heat-shock
treatment. But the gfp mRNA was ubiquitously expressed similarly as
in uninjected embryos (data not shown). These results justified our use of
this AMO for the analysis of loss-of-function phenotypes of
plexinA4.
Staining with anti-acetylated -tubulin antibody revealed that, in
the AMO-injected embryos, the number of the peripheral axons of Rohon-Beard
neurons was reduced as demonstrated in the lateral view
(Fig. 4B,B'). However, as
revealed by the dorsal view (Fig.
4E), the number of the main trunks of the peripheral axons of
Rohon-Beard neurons (arrowheads) exiting the spinal cord was similar as in the
normal embryos (Fig. 4D) and in
the embryos injected with the control MO
(Fig. 4F). This phenotype
caused by injection of AMO partially recapitulated the defects observed in the
embryos that were injected with mRNA for LIMIsl-2, although the
peripheral axons of Rohon-Beard neurons were completely eliminated in the
embryos overexpressing LIMIsl-2
(Fig. 1D').
To analyze the phenotype more precisely, we made the camera lucida drawing and counted the number of the branch termini and branching points of the peripheral axons (Fig. 4A'-C') distributed in each subdivision of the trunk along the dorsoventral axis (D1-D3,V3-V1) (Fig. 4G).
The number of peripheral branches was reduced to about 67.5% compared with wild-type embryos and with the embryos injected with control MO. The dorsoventral distribution of branch termini of the peripheral axons showed slight increase in shorter axons (data not shown). Most conspicuously, the number of branching points was prominently reduced to 42% in the AMO-injected embryos (n=24) (Fig. 4H), especially in the dorsal region (D2 region) where the peripheral axon branches elaborate most extensively in the wild-type embryo (n=8), and in the embryo injected with control MO (n=13) (Fig. 4I).
To further confirm our observation at the single-cell level, we carried out
time-lapse observation of the growth of a single Rohon-Beard neuron in the
embryo ubiquitously overexpressing Slit2-GFP by expressing Kaede in this
neuron (Ando et al., 2002)
(Fig. 5A,B,D,E). Kaede emits
much brighter red signal than DsRed2 in zebrafish embryos when it is
photoconverted by exposure to UV light. The plasmid (pSSICP-Kaede) which can
drive expression of Kaede fluorescent protein under control of the sensory
neuron-specific enhancer of isl1 (SS) was injected into the one-cell
stage normal embryos with AMO against plexinA4. A part of the
embryonic spinal cord expressing Kaede in Rohon-Beard neurons was exposed to
UV light until the emitted light turns from pale blue to red. We confirmed the
reduction in the number of branching points of peripheral axons of a single
Rohon-Beard neuron in the AMO-injected embryo (n=3).
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PlexinA4 is necessary for the Slit signaling in promotion of sensory axon branching
Slit plays a crucial role in axon pathfinding
(Brose et al., 1999;
Li et al., 1999
;
Wang et al., 1999
;
Guthrie, 1999
;
Brose and Tessier-Lavigne,
2000
). In zebrafish, ubiquitous overexpression of Slit2 causes
abnormal axonal pathfinding in the axons of the trigeminal sensory ganglion
neurons and axons of Mauthner neurons, excessive branching of the peripheral
axons of the trigeminal sensory ganglion neurons and Rohon-Beard neurons, and
defasciculation of the medial longitudinal fascicles
(Yeo et al., 2004
) (see also
Fig. 7B,F). We showed that
Robo2 is specifically expressed in the trigeminal sensory neurons, and
disruption of this gene in the mutant embryos cancels the branch promotion
effect of excessive Slit2 overexpression
(Yeo et al., 2004
). Robo3 is
reported to be expressed in the dorsal spinal cord of zebrafish embryos
(Lee et al., 2001
).
The trigeminal sensory ganglion neurons and Rohon-Beard neurons also
express plexinA4, as shown in Fig.
1C,E. The abnormal increase in the number of the peripheral axon
branches of Rohon-Beard neurons in the Slit2-overexpressing embryos
[n=19, 180% increase compared with normal embryos
(n=10)] [Fig. 7B,L,M,
see also Fig. 5C,F
(n=3)] was opposite to the phenotype induced by loss of function of
PlexinA4, as described above (Fig.
4, Fig. 6 and
Fig. 5B,E). Therefore, we
examined whether PlexinA4 and Slit2 might have functional interaction with
each other.
To examine the relationship between Slit2 and PlexinA4, we made transgenic
zebrafish which could overexpress both Slit2-GFP and dnPlexinA4-GFP under
control of the hsp70 promoter
(Yeo et al., 2001). In these
embryos, the excessive branching of the peripheral axons of Rohon-Beard
neurons which would be induced by overexpression of Slit2-GFP alone
(Fig. 7B) was not observed
(Fig. 7C). This effect of
dnPlexinA4 expression is dose dependent. The embryos that carry
SS-hsp70:dnPlexinA4-GFP transgene homozygously showed more prominent reduction
in the number of the branching points (n=20, 64.4%) than did the
embryos carrying the transgene heterozygously (n=20, 109.5%)
(Fig. 7L,M). In addition, when
we injected AMO against plexinA4 into the transgenic embryo
overexpressing Slit2-GFP, the excessive branching was also canceled
(n=8, 82%) (Fig. 7D),
but the Slit2-overexpressing embryo which was injected with control-MO
(n=8, 160%) showed similar excessive branching to the uninjected
embryos (n=8, 176.5%) (Fig.
7N,O).
In normal embryos, the central axons of the trigeminal sensory ganglion
neurons project into the hindbrain as a tight fascicle
(Fig. 7E). Our recent study
revealed that these central axons are extensively defasciculated and fail to
enter the hindbrain in the embryos ubiquitously overexpressing Slit2-GFP under
control of the hsp70 promoter
(Fig. 7F)
(Yeo et al., 2004). By
contrast, in 40% of embryos transgenically overexpressing both Slit2-GFP and
dnPlexinA4-GFP under control of the hsp70 promoter, the central axons
were prevented from defasciculation and entered the hindbrain as in normal
embryos (Fig. 7G;
Table 1). Similar results were
obtained in 44% of the embryos overexpressing Slit2-GFP when they were also
injected with AMO for plexinA4
(Fig. 7H;
Table 1).
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These results indicate that PlexinA4 interacts with the Slit2 signaling specifically to promote axonal branching of Rohon-Beard neurons and the trigeminal sensory ganglion neurons.
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Discussion |
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In the embryos overexpressing LIMIsl-2, the peripheral axons of
the primary sensory neurons were completely eliminated, while their central
axons remained intact (Segawa et al.,
2001). In contrast to this distinctive effects of functional
repression of Islet2, loss of PlexinA4 function induced more limited
abnormality, i.e. reduction in axonal branching without severely impairing
axonal extension. Some other genes under control of Islet2 may be regulating
extension of the peripheral axons of the primary sensory neurons in zebrafish
embryos. In fact, expression level of mRNA for other genes such as
pea3 (M. Mieda and H.O., unpublished) and TrkC1 (H.S. and
H.O., unpublished) in the primary sensory neurons are also reduced by
overexpression of LIMIsl-2.
PlexinA4 functions in axonal branching
Proteins of the PlexinA family form a co-receptor complex with neuropilin
for their ligand, class III secreted semaphorins and function as its signal
transducer (Takahashi et al.,
1999; Tamagnone et al.,
1999
; Rohm et al.,
2000
). Semaphorins are known to induce collapse and repulsion of
the growth cone of cultured DRG neurons. In this study, we demonstrated that
zebrafish plexinA4 is a downstream target gene of a
LIM-homeodomain-type transcription factor Islet2 and is involved in
development of primary sensory neurons especially in branching of the
peripheral axons in the primary sensory neurons, rather than mediating the
repulsive signaling. In Drosophila, PlexA interacts with Sema1a, and
promotes branching of the axons of SNa motoneuron
(Winberg et al., 1998
). This
suggests that Plexin function for promotion of axonal branching is
evolutionarily conserved. At this point, we have no evidence as to whether the
branch-promoting activity of PlexinA4 in the zebrafish primary sensory neurons
required semaphorins. In zebrafish, several semaphorin genes were cloned
(Halloran et al., 1998
;
Halloran et al., 1999
;
Yee et al., 1999
;
Roos et al., 1999
). One of the
secreted Sema, Sema3d, is expressed in roof plate cells at dorsal
midline (Halloran et al.,
1999
). It would be intriguing to examine its functional
relationship to PlexinA4.
Role of PlexinA4 in the Slit signaling
Slit is also known to promote axonal branching of the NGF-responsive
neurons in the DRG (Wang et al.,
1999). In addition, ubiquitous overexpression of Slit2-GFP in the
transgenic zebrafish embryos by heat-shock treatment increased the number of
branching of the peripheral axons from the primary sensory neurons such as
Rohon-Beard neurons and the trigeminal sensory ganglion neurons
(Yeo et al., 2004
). Loss of
function of PlexinA4 not only reduced the number of branches of the peripheral
axons from Rohon-Beard neurons but also counteracted the effects of
overexpression of Slit2-GFP on axonal branching. These results suggested that
PlexinA4 is necessary for promotion of branching of the peripheral axons of
both Rohon-Beard neurons and the trigeminal sensory ganglion neurons by the
Slit signaling cascade.
Little is known about why different neurons respond to Slit in different
ways. Slit2 is known to be proteolytically processed into a 140 kDa N-terminal
fragment and a 55-60 kDa C-terminal fragment in vivo. In addition, the
full-length fragment is also found from rat brain extracts. The N-terminal
fragment of Slit2 (Slit2-N) but not the full-length Slit2 induces branching
(Wang et al., 1999;
Nguyen Ba-Charvet et al.,
2001
). The uncleaved form of Slit2 (Slit2-U) instead acts as an
antagonist for Slit2-N in promotion of branching
(Nguyen Ba-Charvet et al.,
2001
).
In contrast to the excessive branching of the peripheral axons of the
primary sensory neurons, and defasciculation and deflection of the central
axons of the trigeminal sensory ganglion neurons, overexpression of
dnPlexinA4-GFP could not rescue the projection errors of Mauthner neurons,
which are induced by overexpression of Slit2-GFP
(Fig. 7K)
(Yeo et al., 2004). Although
Mauthner neurons also express both plexinA4 and robo3
(Yeo et al., 2004
), they start
expressing plexinA4 much later (
21-22 hpf) after their growth
cones have already crossed the midline (15-16 hpf). These results indicated
that function of PlexinA4 is not involved in collapse or repulsion of the
growth cones of the Mauthner neurons at the midline of hindbrain in response
to the Slit signals. Therefore, colocalized expression of Robo
(Lee et al., 2001
;
Yeo et al., 2004
), a receptor
for Slit2, and PlexinA4 may be essential only for promotion of axonal
branching in response to Slit but not for the growth cone collapse.
Colocalization of the signaling cascades mediated by Slit and class III
semaphorins is also observed in the dendrites of the pyramidal neurons of the
cortex of the mammals. Slit1 acts as a chemorepellent for the axons of these
neurons. By contrast, it induces dendritic growth and branching. This
dendritic growth and branching is dependent on Slit-Robo system
(Whitford et al., 2002).
Sema3A act as a chemorepellant for axon and a chemoattractant for apical
dendrites. These differences arise from asymmetric cellular localization of
soluble guanylate cyclase. Soluble guanylate cyclase is asymmetrically
localized to the dendrites, causing higher concentration of cGMP in dendrites
than in axons (Polleux et al.,
2000
). Interaction of Slit and Sema3 signaling may be important
for the modeling of the dendrites of cortical pyramidal cells in mammals.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Laboratory of Molecular Genetics, NICHD, NIH, 20892,
Bethesda, MD, USA
Deceased and to whom this paper is dedicated
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