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First published online 30 November 2005
doi: 10.1242/dev.02187
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1 Division of Biology 139-74, California Institute of Technology, Pasadena, CA
91125, USA.
2 Department of Neuroscience, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205, USA.
3 Howard Hughes Medical Institute, The Johns Hopkins University School of
Medicine, Baltimore, MD 21205, USA.
* Author for correspondence (e-mail: mbronner{at}caltech.edu)
Accepted 27 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Trunk neural crest migration, Sclerotome, Neuropilin 2, Semaphorin 3F, Mouse, Chick
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Rickman et al., 1985;
Serbedzija et al., 1990). This
selective migration results in the formation of segmentally arranged streams
of migrating neural crest cells. This pattern appears to be imposed by the
somites, with the anterior sclerotome being permissive and posterior
sclerotome repulsive for neural crest migration
(Bronner-Fraser and Stern,
1991; Kalcheim and Teillet,
1989). Accordingly, surgical or genetic modification of
anteroposterior somite polarity results in loss of segmental neural crest
migration (Kalcheim and Teillet,
1989) and formation of fused neural crest-derived dorsal root
ganglia (Bussen et al., 2004;
Kalcheim and Teillet, 1989;
Leitges et al., 2000;
Mansouri et al., 2000). Thus,
the segmental pattern of neural crest migration is believed to be responsible
for the metameric organization of the ganglia of the peripheral nervous system
(Kuan et al., 2004;
LeDouarin and Kalcheim, 1999).
The segmented arrangement of these ganglia relative to the somites, which will
form the vertebrae, is crucial for proper wiring of the ganglia and peripheral
nerves to targets in the periphery.
The identity of the molecular cues that direct neural crest migration
exclusively through the anterior sclerotome is still open to debate. Although
previous reports suggested that Eph/ephrin signaling might pattern trunk
neural crest migration (Krull et al.,
1997; Wang and Anderson,
1997), the Eph and ephrin mutant mice that have been
examined fail to exhibit trunk neural crest migration defects
(Adams et al., 2001;
Davy et al., 2004;
Orioli et al., 1996;
Wang et al., 1998). Likewise,
neuropilin 1 and its ligand semaphorin 3A have been suggested to play a role
(Eickholt et al., 1999), but
are not expressed at the right time (reviewed by
Kuan et al., 2004) and are not
required in the mouse for appropriate trunk neural crest migration
(Kawasaki et al., 2002). It is
not clear whether the inability to identify a trunk neural crest mutant
phenotype is due to redundancy or whether the true regulatory molecules have
not been found.
We isolated chick neuropilin 2a1 (Npn2a1) in a screen for genes
upregulated as a consequence of neural crest induction
(Gammill and Bronner-Fraser,
2002). Npn2 is a receptor for class 3 secreted semaphorins (Sema)
3C and 3F as well as vascular endothelial growth factor
(Bagri and Tessier-Lavigne,
2002; Neufeld et al.,
2002). Npn2 is required for appropriate axon guidance and
fasciculation in the central and peripheral nervous system
(Chen et al., 2000;
Cloutier et al., 2002;
Giger et al., 2000). However,
the importance of Npn2 and its ligands during trunk neural crest development
has not been examined.
Here, we explore the role of Npn2 signaling during neural crest migration. We demonstrate that the Npn2 receptor on neural crest cells detects a Sema3f repellant cue in the posterior sclerotome that guides neural crest migration through the somites. Surprisingly, individualized dorsal root ganglia still form, albeit less well separated than normal, suggesting that the pattern of neural crest migration alone does not dictate the arrangement of the peripheral ganglia, and that multiple signaling pathways are required to create a segmented peripheral nervous system.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Embryos were isolated in Ringers Saline and fixed overnight
at 4°C in 4% paraformaldehyde. Mouse embryos were surgically isolated,
with day 0.5 being the day of the plug, into ice cold phosphate-buffered
saline (PBS) and fixed for 2 hours at room temperature or overnight at 4°C
in 4% paraformaldehyde. Embryos were genotyped by polymerase chain reaction
(PCR) (Giger et al.,
2000).
In situ hybridization
Chick in situ hybridization was performed as described previously
(Gammill and Bronner-Fraser,
2002). Mouse in situ hybridization was performed as described
previously (Wilkinson, 1992),
except that hybridization was performed in 50% formamide, 1.3x SSC (pH
5), 5 mM EDTA, 50 µg/ml yeast RNA, 0.2% Tween 20, 0.5% CHAPS and 50
µg/ml heparin at 70°C. Embryos were washed twice in hybridization mix
at 70°C, three times in wash solution I at 65°C, and antibody
pre-treatment was performed in 100 mM maleic acid, 150 mM NaCl, 0.1% Tween (pH
7.5) with 2% Blocking Reagent (Boehringer Mannheim). Templates for
digoxigenin-labeled antisense riboprobes were as follows: chick Npn2
(Gammill and Bronner-Fraser,
2002), mouse Npn2
(Giger et al., 2000),
Sox10 (Kuhlbrodt et al.,
1998), Sema3f (Giger
et al., 2000), ephrinB2 (Wang
and Anderson, 1997), Tbx18
(Kraus et al., 2001) and
Uncx4.1 (Mansouri et al.,
1997). Stained embryos were infiltrated with 5% sucrose, 15%
sucrose and 7.5% gelatin in 15% sucrose, frozen in liquid nitrogen, sectioned
at 20 µM by cryostat (Microm) and mounted in permafluor (Thermo Electron
Corporation).
Immunohistochemistry
Neural crest cells with were stained with 1:50 anti-HNK-1 (American Type
Culture; Tucker et al., 1984)
followed by 1:400 anti-mouse-IgM-Rhodamine Red X (Jackson Immuno Research) or
1:2000 anti-p75 (Weskamp and Reichardt,
1991) followed by 1:400 anti-rabbit-Rhodamine Red X (Jackson
Immuno Research). Sema3f spots were visualized using an anti-mouse IgG Alexa
488 secondary at 1:1000 (Molecular Probes). Unstained embryos were infiltrated
with 5% sucrose, 15% sucrose and 7.5% gelatin in 15% sucrose, frozen in liquid
nitrogen, sectioned at 15 µM by cryostat (Microm) and degelatinized for 20
minutes at 42°C in PBS. Dorsal root ganglia were stained with 1:500
anti-TUJ1 (neuron specific class III ß-tubulin; Babco) followed by 1:500
anti-mouse-Biotin (Jackson Immuno Research), and developed using the
ABC-horseradish peroxidase kit (Vector Laboratories) and 0.1 mg/ml
3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) with 0.009%
hydrogen peroxide according to the manufacturer's instructions.
Conditioned medium
293T cells were transfected with 24 µg of AP-Sema3f
(Giger et al., 2000) or AP
empty vector using 45 µl Lipofectamine 2000 (Invitrogen) per 10 cm dish
according to the manufacturer's instructions. Media (DMEM + 0.1% BSA + Pen
Strep) was collected after 3 days and concentrated using a Centriplus YM-100
filter device (Millipore). Alkaline phosphatase activity was determined using
AP Assay Reagent A (GenHunter Corporation) and the molarity of the collected
protein calculated according to the manufacturer's instructions.
Sema3f spot preparation
Aminopropyltriethoxysilane (2%; APTES; Sigma) was prepared in 95% ethanol
and allowed to hydrolyze for 5 minutes in a fume hood. Thermanox (25 mm; Nunc)
cover slips in a wafer basket (Fluoroware) were incubated in the 2% APTES for
10 minutes, and washed three times for 5 minutes with 95% ethanol. Coverslips
were cured for 15 minutes at 100°C in a vacuum oven, immobilized onto 35
mm tissue culture plates with four spots of silicone vacuum grease, and UV
sterilized in a tissue culture hood for 15 minutes. AP-Sema3f (75 nM) was
preincubated at room temperature for 1 hour with 50 µg/ml mouse anti-human
placental alkaline phosphatase (Chemicon), 0.2 µl drops were spotted
manually in a grid on the coverslip, and the location of the spots was marked
on the underside of the dish. After 1 hour at 37°C, the coverslips were
washed three times with 4 ml of 1x Hank's buffered saline solution
(HBSS; Invitrogen). After all remaining traces of HBSS had been aspirated,
150-200 µl of 125 µg/ml fibronectin (BD Biosciences) was laid over the
spots and incubated for 1.5 to 2 hours at 37°C. After aspirating the
fibronectin, the coverslips were washed once with 4 ml of HBSS and stored
overnight at 4°C in 2 ml DMEM-F12 (Invitrogen) + 1 mg/ml BSA.
Mouse neural tube culture
E9.5 embryos (14-24 somites) were isolated into ice cold HBSS. The region
of the trunk containing the last 10 somites was dissected, trimming the
membranes lateral to the somites and removing the gut tube. Trunk pieces were
incubated for 8 minutes at 37°C in room temperature 3 µg/ml dispase
made fresh in HBSS and 0.2 µm filter sterilized. After rinsing several
times with DMEM-F12 + 10% fetal bovine serum (Hyclone), neural tubes were
isolated by trituration with a fire-polished pasteur pipette and plated on the
spotted region of the coverslips in 1 ml of pre-warmed neural crest complete
medium, prepared as described (Stemple and
Anderson, 1992) except that DMEM-F12 was used and retinoic acid
was omitted. The tubes were allowed to stick to the coverslip for one hour at
37°C, then 2 ml of additional complete medium was added slowly down the
side of the dish, and explants were cultured for an additional 28-48 hours at
37°C.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Npn2 is expressed in premigratory neural crest
cells in the dorsal neural tube as well as on migratory neural crest in both
the chick (Fig. 1A,B)
(Gammill and Bronner-Fraser,
2002) and the mouse (Fig.
1C-F). In the trunk, Npn2 was clearly expressed in a
segmental pattern in both species. Longitudinal sections through chick embryos
revealed that Npn2 was expressed by neural crest cells, identified by
HNK-1 immunoreactivity, as they migrate through the anterior half of each
somitic sclerotome (Fig. 1B).
Similarly, in transverse sections of mouse embryos, Npn2 expression
colocalized with the neural crest marker p75
(Fig. 1D-F).
The distribution of Npn2 on neural crest cells made this receptor
a potential candidate for influencing neural crest formation and migration. To
address the requirement for Npn2 during neural crest development, we assessed
the loss-of-function phenotype by characterizing neural crest marker gene
expression in Npn2-null mutant mice
(Giger et al., 2000). In situ
hybridization with probes for the neural crest markers Sox10, Foxd3
and Pax3 at E8-8.5 (4 to 12 somites) showed no obvious effects on the
specification or generation of neural crest cells in wild-type, heterozygous
or Npn2 mutant mice (data not shown).
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). In
Npn2 mutant mice, however, neural crest cells migrated in a uniform
sheet rather than in streams (Fig.
2D), and Sox10-positive cells were present throughout
both anterior and posterior portions of the sclerotome
(Fig. 2E,F). More ventrally,
approaching the dorsal aorta where the neural crest-derived sympathetic
ganglia will form, some segmental neural crest migration was still apparent in
Npn2-null mice (Fig.
2D). Interestingly, motor axons, which normally are restricted to
the anterior sclerotome, also project into both anterior and posterior
sclerotome in Npn2 mutants, although ventral roots still form (data
not shown). Together, these data suggest that Npn2 signaling is required for
neural crest guidance events through dorsal and intermediate levels of the
somite.
Npn2 and Sema3f exhibit complementary expression patterns
Npn2 can bind to three different ligands: Sema3C, Sema3f and vascular
endothelial growth factor (Chen et al.,
1997; Gluzman-Poltorak et al.,
2000). To determine which ligand was mediating the patterning
functions revealed by the Npn2 mutant, we next assessed the
expression patterns of these molecules by in situ hybridization and
immunohistochemistry. Sema3c is first expressed in the somites around
E10, and then only in the dermomyotomal compartment
(Adams et al., 1996).
Immunostaining for vascular endothelial growth factor was also detected at low
levels in the dermomyotome but not in the sclerotome at E9.5 (data not shown).
By contrast, Sema3f was expressed in a pattern complementary to that
of Npn2 (Fig. 3A,B),
with Npn2 expression in the anterior somite
(Fig. 3C) mirrored by
Sema3f expression in the posterior somite
(Fig. 3D). In sections,
Npn2 was clearly expressed in the anterior half of each sclerotome
(Fig. 4A), and Sema3fF
in the posterior half (Fig.
4B). This expression pattern made Sema3f an ideal
candidate for signaling through the Npn2 receptor during trunk neural
crest migration. The complementary distribution of Npn2 and
Sema3f expression has also been observed at later stages of
development (Giger et al.,
2000; Giger et al.,
1998).
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).
Strikingly, the Sema3f-null neural crest migration phenotype was
identical to that observed in Npn2 mutants. Instead of segmentally
arranged streams of neural crest cells in the trunk
(Fig. 3E), Sema3f
nulls contained uniform sheets of migrating neural crest
(Fig. 3G). In sections,
alternating Sox10-positive and - negative regions were observed in
the anterior- and posterior-half sclerotome, respectively, of wild-type mice
(Fig. 3F), whereas uniformly
distributed Sox10-labeled cells were seen throughout the sclerotome
of Sema3f mutants (Fig.
3H). Together, these results demonstrate that signaling between
the receptor Npn2 and its ligand Sema3f is required to restrict neural crest
migration to the anterior somite.
Somite patterning is normal in Npn2 and Sema3f mutants
Two alternate mechanisms could explain the trunk neural crest migration
defects observed in the Npn2 (Fig.
2) and Sema3f (Fig.
3) mutant mice. The phenotype could reflect a requirement for
signaling between the Sema3f repulsive ligand in the posterior somite and the
Npn2 receptor on neural crest cells to guide neural crest migration.
Alternatively, Npn2/Sema3f signaling between the anterior and posterior somite
could be important for maintenance of anterior and/or posterior sclerotomal
identity. Disrupting this signaling could affect somite polarity and thus the
environment through which neural crest migrates, secondarily impacting the
pattern of neural crest migration. For example, anteroposterior somite
polarity is abolished in Delta1 mutant mice
(deAngelis et al., 1997), and
as a consequence, neural crest cells migrate aberrantly through the posterior
sclerotome of these animals (DeBellard et
al., 2002).
To differentiate between these two possibilities, markers of anterior and
posterior sclerotome were examined in wild-type, Npn2 mutant and
Sema3f mutant mice to determine whether anterior and posterior somite
identity was retained in the mutants. Sema3f was expressed in the
posterior sclerotome of both wild-type
(Fig. 4B) and Npn2
mutant mice (Fig. 4C). Ephrin
B2, a ligand that repels migrating neural crest cells in vitro
(Wang and Anderson, 1997), was
also equivalently restricted to the posterior sclerotome of wild-type
(Fig. 4D) and Npn2
mutant embryos (Fig. 4E). Thus,
two posteriorly expressed guidance molecules are appropriately localized in
Npn2 mutants.
Somite polarity is established during segmentation of the somitic mesoderm,
with anterior and posterior somite identity maintained and promoted by two
different transcription factors, Tbx18
(Bussen et al., 2004) and
Uncx4.1 (Leitges et al., 2000;
Mansouri et al., 2000).
Tbx18 was restricted to the anterior sclerotome of wild-type
(Fig. 4F) and Npn2
mutant embryos (Fig. 4G).
Uncx4.1 was also properly expressed in the posterior sclerotome of
wild-type (Fig. 4H),
Npn2 mutant (Fig. 4I)
and Sema3f mutant mice (Fig.
4J). These results demonstrate that anterior and posterior
sclerotomal character is maintained in Npn2 and Sema3f
mutants, suggesting that the requirement for Npn2/Sema3f signaling is likely
to reside in the neural crest.
Npn2 is required in the neural crest for Sema3f-mediated repulsion
To test the requirement for Npn2 in the neural crest directly, we explanted
wild-type and Npn2 mutant neural tubes, and cultured them on
fibronectin-coated substrates containing spots of Sema3f. Wild-type neural
crest cells avoided Sema3f (Fig.
5A), with the majority of cells remaining at the spot border and
only individual, rare cells migrating onto the Sema3f substrate, consistent
with their behavior in vivo
(Kasemeier-Kulesa et al.,
2004). By contrast, Npn2 mutant neural crest cells
migrated equally well on fibronectin with or without Sema3f protein
(Fig. 5B,C). This demonstrates
that the Npn2 receptor on neural crest cells detects a Sema3f repulsive cue in
the environment, and supports a cell-autonomous requirement for Npn2 on the
neural crest during trunk neural crest migration.
Segmentally arranged dorsal root ganglia form in Npn2 mutant mice
The segmental migration of neural crest through the somite is thought to
prefigure the segmented organization of the neural crest-derived ganglia of
the peripheral nervous system. This pattern ensures that the ganglia and the
vertebrae, which differentiate from the somites, will form in register with
one another. For example, in each somite, neural crest cells in the anterior
sclerotome coalesce to form the dorsal root ganglia. As a result, in
Npn2 mutants, one might expect a continuous mass of dorsal root
ganglia to form instead of individualized ganglia. This is the case when
neural crest migrates non-segmentally through somites that are genetically
(deAngelis et al., 1997;
DeBellard et al., 2002) or
surgically manipulated (Kalcheim and
Teillet, 1989) to contain only anterior character. In wild-type
embryos at E10.5 and E11.5, the streams of Sox10-expressing neural
crest cells in the trunk condensed into ganglia in an anterior to posterior
progression (Fig. 6A,C).
Strikingly, neural crest cells in Npn2 mutant mice also coalesced
into recognizable ganglia. At E10.5, neural crest cells were still distributed
throughout the somites, but became excluded from the somite boundaries
(Fig. 6B). By E11.5,
individualized ganglia appeared to have sorted out from the sheet of migrating
neural crest in the somite (Fig.
6D, arrowheads mark the same axial levels in all panels). In
longitudinal sections at E11.5, TUJ1 immunoreactivity confirmed the apparent
segmentation, with space between each Npn2 mutant dorsal root
ganglion (Fig. 6F). Although
morphologically similar to those in wild-type embryos
(Fig. 6E), the mutant ganglia
were not as well separated, suggesting that the process of gangliogenesis
occurred but was somewhat compromised. The sympathetic ganglia, which form in
a segmental pattern ventral to the somites, are normal in Npn2
mutants (Giger et al.,
2000).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Npn2/Sema3f signaling patterns neural crest migration in the trunk
In Npn2 and Sema3f mutant mice, trunk neural crest cells
migrate through both the anterior and posterior sclerotome, rather than
exclusively through the anterior-half sclerotome as in wild-type mice. This
demonstrates that signaling between the receptor Npn2 and its ligand Sema3f is
required to restrict neural crest migration to the anterior somite.
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), where neural crest migration has a pseudo-segmental
appearance, with migrating neural crest cells present throughout the
sclerotome but avoiding somite boundaries
(DeBellard et al., 2002). This
comparison suggests that, unlike Delta1, the Npn2 defect is
not in the somites themselves. Third, as Npn2 is expressed on
anterior sclerotomal cells, if Npn2/Sema3f were involved in somite patterning,
loss of signaling through this receptor would most probably posteriorize the
sclerotome. A posteriorized somite impedes neural crest migration, leading to
reduced numbers of migratory neural crest cells and their derivatives
(Bussen et al., 2004;
Kalcheim and Teillet, 1989).
However, this is not the case in Npn2-null mice. For these reasons,
we favor the idea that Npn2 is required in the neural crest.
What is the purpose, then, of Npn2 expression in both the neural
crest and the sclerotome through which it migrates? Interestingly, like
Npn2, Npn1 (Eickholt et al.,
1999) and Ephb3 receptors
(Krull et al., 1997), both of
which have been postulated to play a role in patterning trunk neural crest
migration, are also distributed on both neural crest and anterior sclerotomal
cells. One intriguing possibility is that these receptors do not play a
signaling role in the somite, but rather serve as a sink to bind up any
repulsive ligand diffusing from the posterior sclerotome, thus ensuring a
sharp boundary such that the anterior sclerotome is devoid of the repulsive
cue.
This is the first report of a single receptor/ligand pair that is
absolutely required to pattern trunk neural crest migration. The molecular
basis for segmental neural crest migration has preoccupied this field since
the phenomenon was first observed. Many different cell adhesion molecules,
extracellular matrix molecules and receptor/ligand pairs have been identified
that are expressed in anterior or posterior sclerotome or in the neural crest,
and in some cases they have been shown to be sufficient to direct neural crest
migration (Kuan et al., 2004).
But in no case has a requirement for any molecule been previously demonstrated
in the embryo. Other signals, such as Eph receptor/ephrin ligand interactions,
might fine tune neural crest migration, or in the case of Npn1/Sema3A, be
involved in later steps in the process. However, Npn2/Sema3f signaling is
clearly the key determinant patterning anterior-only migration through the
sclerotome.
Segmental neural crest migration may not be required for segmental dorsal root ganglion formation
The requirement for Npn2/Sema3f signaling in neural crest migration has
uncovered an additional, previously unrecognized process that results in
dorsal root ganglion segmentation irrespective of the neural crest migration
pattern. Despite the fact that neural crest cells migrate through the anterior
and posterior sclerotome of Npn2 mutant mice, segmentally arranged
dorsal root ganglia still form. This result suggests that segmental neural
crest migration and subsequent sequestration of ganglia are separable events.
One explanation is that there may be independent signals restricting neural
crest migration and the pattern of ganglion aggregation. For example, either a
cell-adhesive, `sorting' signal or a repulsive cue in the posterior sclerotome
could promote aggregation within the anterior sclerotome, irrespective of the
starting location of the neural crest cells within the sclerotome. In favor of
this possibility, fused dorsal root ganglia form when anteroposterior somite
polarity is abolished by either surgical or genetic manipulation
(Bussen et al., 2004;
Kalcheim and Teillet, 1989;
Leitges et al., 2000;
Mansouri et al., 2000). This
indicates that anteroposteriorly patterned signals in the somite are required
for segmental formation of dorsal root ganglia. Candidates for such signals
include F-spondin (Debby-Brafman et al.,
1999) and Npn1 (Kitsukawa et
al., 1997).
A second possibility is that dorsal root ganglia form in the absence of
segmental neural crest migration simply because neurons tend to aggregate (M.
Bronner-Fraser, unpublished). In support of this stochastic mechanism, when
normal somites are surgically replaced with multiple anterior or posterior
somite halves, a giant mass of ganglia forms that exhibits a pseudo-segmental
appearance, with alternating thick and thin regions at random intervals within
the giant ganglion (Kalcheim and Teillet,
1989). That they are fused, however, argues that a combination of
these two mechanisms is normally at play.
Finally, it is also possible that the physical structure of the somite
itself can impose segmentation during gangliogenesis. In addition to
regionally restricted molecular cues, such as Sema3f expression
posteriorly, there are embryological boundaries and differences within the
sclerotome. These include, most notably, the intersomitic space, as well as
von Ebner's fissure between anterior and posterior sclerotome, and the various
subdomains within the sclerotome (reviewed by
Christ et al., 2004). Anterior
sclerotome is less cell dense than posterior sclerotome
(Christ et al., 2004), is
mitogenic for dorsal root ganglia
(Goldstein et al., 1990) and
will undergo apoptosis in the absence of neural crest cells (see
Christ et al., 2004). All of
these segmentally restricted differences could have a morphological impact
during gangliogenesis. In the case of the Npn2 mutants, the uniform
sheet of migrating neural crest cells appears to segment into individual
dorsal root ganglia at the somite border
(Fig. 6). Interestingly, when
chick embryos are surgically modified to contain only anterior sclerotome, in
other words have no somite boundary, neural crest migrates non-segmentally and
dorsal root ganglia are fused (Kalcheim
and Teillet, 1989). However, dorsal root ganglia are also fused in
Uncx4.1 and Tbx18 mutants, where anteroposterior somite
polarity is abolished but physical somites still form
(Bussen et al., 2004;
Leitges et al., 2000;
Mansouri et al., 2000).
Together, these data suggest that somite polarity creates positional
information at the somite boundary that impacts upon the segmentation of the
peripheral nervous system. Migrating neural crest cells normally maintain
filopodial contact across the posterior somite and can even cross over between
adjacent streams (Kasemeier-Kulesa et al.,
2004), thus the somite boundary could normally curtail this
movement as dorsal root ganglia condense.
The sympathetic ganglia also are not dependent upon the pattern of neural
crest migration for their segmented organization. By imaging actively
migrating neural crest cells, Kasemeier-Kulesa and colleagues
(Kasemeier-Kulesa et al.,
2004) showed that, once they have passed through the somites,
neural crest cells no longer maintain their segmental position and can migrate
as far as two segments anteriorly or posteriorly. The mechanisms that
ultimately result in the aggregation of these cells into individualized
sympathetic ganglia are likely to be similar to those we propose for the
formation of metameric dorsal root ganglia in Npn2 mutants.
Interestingly, Npn1 and Sema3A are required for localization and condensation
of sympathetic precursors as well
(Kawasaki et al., 2002).
The overriding message is that the segmental migration of neural crest
cells through the somites itself is not requisite for the creation of a
segmented peripheral nervous system, despite what has been assumed for 20
years (reviewed by Kuan et al.,
2004). Although the dorsal root ganglia eventually segment in the
Npn2 mutants, they are more closely spaced than normal. Thus, the
pattern of neural crest migration is important, but not essential for the
formation of segmented dorsal root ganglia. This may not be surprising given
the regulative nature of vertebrate embryos, which may have `back-up'
mechanisms for formation of important organ systems in the event that primary
mechanisms are perturbed.
Functional validation of the neural crest gene expression profile
We originally identified Npn2a1 in a screen for genes upregulated
in response to neural crest induction
(Gammill and Bronner-Fraser,
2002). Our current analysis of Npn2 function has several
implications. First of all, the importance of Npn2 for neural crest migration
validates our neural crest gene expression profile and demonstrates that our
collection of genes contains true regulators of neural crest development. This
conclusion is supported by the demonstration that Laminin-
5, another
gene identified in our screen, is also important for proper emigration of
neural crest cells (Coles et al.,
2005).
In addition, although we screened for genes expressed in premigratory
neural crest (Gammill and Bronner-Fraser,
2002), Npn2 is required for neural crest migration and apparently
not for specification, as no differences were noted in the expression of early
neural crest markers Sox10, Pax3 and FoxD3 in the neural
folds and dorsal neural tube of the mouse. We cannot, however, rule out the
possibility of an early role for chick Npn2 in neural crest specification, as
it is expressed earlier and at higher levels in this organism. Regardless,
genes crucial for migration are clearly expressed in premigratory neural crest
as a consequence of neural crest induction. This supports our model that early
neural crest development entails a sequential activation of migratory
potential, with a signal to migrate activating this potential in a subset of
premigratory neural crest cells (Gammill
and Bronner-Fraser, 2002). Further analysis of our neural crest
gene collection promises to reveal the roles of additional genes in this
process.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adams, R., Diella, F., Hennig, S., Helmbacher, F., Deutsch, U. and Klein, R. (2001). The cytoplasmic domain of the ligand EphrinB2 is required for vascular morphogenesis but not cranial neural crest migration. Cell 104,57 -69.[CrossRef][Medline]
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