Department of Molecular Biology and Genetics, 445 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA
* Author for correspondence (e-mail: kew13{at}cornell.edu)
Accepted 11 October 2005
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SUMMARY |
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Key words: Midbrain, GnRH2, Kallmann Syndrome, Neural crest, Morpholinos
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Introduction |
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The vertebrate head arises through a complex interplay between epithelial
and mesenchymal cell populations. Prominent among the latter are cells derived
from the cranial neural crest. Depending on the specific structure, neural
crest cells may be the sole contributor or may complement contributions from
other organs, e.g. mesoderm and placodes
(Hall, 1999). Cranial neural
crest cells give rise to a myriad of different cell types, including but not
limited to: neurons, glia, pigment, and many skeletal elements of the head.
Cranial neural crest cells also contribute to the specialized sensory systems,
which have become concentrated and elaborated as the structure of the
vertebrate head evolved (Le Douarin and
Kalcheim, 1999
).
Neural crest development is a complex, multi-step process, involving a
myriad of inducing signals and responding transcription factors
(Heeg-Truesdell and LaBonne,
2004). Two genes encoding transcription factors important for
neural crest differentiation are foxd3 and sox10. A member
of the forkhead family of transcription factors, foxd3 is expressed
in neural crest precursor cells. In Xenopus, overexpression of
foxd3 results in the induction of neural crest-specific genes and
loss of function suppressed the expression of neural crest markers, suggesting
a primary role in neural crest cell differentiation
(Sasai et al., 2001
).
Zebrafish foxd3 is expressed in the pre-migratory neural crest,
floorplate, somites and tailbud (Odenthal
and Nusslein-Volhard, 1998
). Subsequently, it has also been shown
to be expressed in glia of the peripheral nervous system
(Kelsh et al., 2000
). The
Sry-related transcription factor gene sox10 is expressed in
pre-migratory neural crest, and loss-of-function studies support its role in
promoting the survival of undifferentiated neural crest cells
(Honore et al., 2003
;
Mollaaghababa and Pavan,
2003
). In zebrafish, sox10 is expressed in developing
pre-migratory neural crest and plays an important role in specifying
non-ectomesenchymal (neurons, glia and pigment) neural crest
(Dutton et al., 2001b
); in
mammals, it is important for the maintenance of multipotency in neural crest
stem cells (Kim et al.,
2003
).
Deficits in GnRH (hypogonadic hypogonadism) underlie some human disease.
Kallmann Syndrome in humans is characterized by deficits in GnRH that are
associated with anosmia (loss of sense of smell). Previous studies have shown
that development of the terminal nerve and hypothalamic GnRH cells is
disrupted in a human Kallmann embryo
(Schwanzel-Fukuda et al.,
1989), and that this phenotype results from mutations in the KAL
(anosmin 1) gene (Duke et al.,
1995
; Legouis et al.,
1991
). Homologues of the kallmann gene have yet to be
identified in mouse, but, in the zebrafish, there are two kallmann
genes, kallmann 1.1 and kallmann 1.2 (Kallman syndrome
1a and Kallman syndrome 1b - Zebrafish Information Network). The
genes are widely expressed in regions of the developing central and peripheral
nervous system, as well as in the reproductive tract and pronephric ducts
(Ardouin et al., 2000
).
The function of midbrain GnRH2 cells suggested that, like the GnRH cells of the terminal nerve, they may arise from the cranial neural crest. We report that the decrement of foxd3 and sox10 gene function results in the loss of midbrain GnRH2 cells, as well as the loss of terminal nerve GnRH3 cells. In addition, we show, using a sox10-GFP line, that the GFP signal and gnrh2 expression co-localize during a discreet time window in early development, thereby supporting a neural crest origin for these cells. Finally, we demonstrate that the decrement of function of the kallmann 1.1 gene, but not the kallmann 1.2 gene, results in loss of the hypothalamic endocrine GnRH1 cells, but did not affect the neuromodulatory midbrain or terminal nerve GnRH cells. Together, these data suggest that the neuromodulatory midbrain GnRH2 cells, like the terminal nerve GnRH cells, have a neural crest origin.
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Materials and methods |
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Morpholinos
Morpholinos (MOs), modified oligonucleotides that interfere with mRNA
translation (Heasman, 2002;
Nasevicius and Ekker, 2000
),
have successfully been used in zebrafish and Xenopus to reduce gene
function, including that of genes important for neural crest development
(Dutton et al., 2001a
;
Honore et al., 2003
;
O'Brien et al., 2004
). Here,
we used MOs to knockdown the function of the foxd3, sox10, kal1.1 and
kal1.2 genes. All MOs and corresponding 5-nucleotide mismatch
controls (mm; mismatches indicated in lower case in sequence below) were
synthesized by the manufacturer (Gene Tools, OR), using existing sequence
information as follows:
foxd3 MO, 5'-CACTGGTGCCTCCAGACAGGGTCAT-3';
foxd3 mm, 5'-CAgTGcTGCCTgCAGACAGcGTgAT-3' (GenBank #NM131290);
sox10 MO, 5'-ATGCTGTGCTCCTCCGCCGACATCG-3';
sox10 mm 5'-ATcCTcTGCTCaTCCGaCGAgATCG-3' (GenBank #AF402677);
kal1.1 MO, 5'-CCGTCGCGCATCTTGAAGAACAGTA-3';
kal1.1 mm, 5'-CCcTCcCGCATgTTGAAaAACAcTA-3' (GenBank #AF163310);
kal1.2 MO, 5'-GCAGAGATTCCTCAAAAGCAGCATC-3';
kal1.2 mm, 5'-GCAGAcATTgCTgAAAAGgAGgATC-3' (GenBank #AF163311).
The foxd3 and kal1.1 MOs were tagged with lissamine (red
fluorescence), and the sox10 and kal1.2 MOs were tagged with
fluorescein (green fluorescence). This allowed for visualization of the MOs in
the embryos. The control mismatch MOs were co-injected with rhodamine dextran
dye (Molecular Probes), at a final concentration of 0.02%. For each MO and
mismatch MO, 1, 5 and 10 nl of MO were tested to determine the most effective
concentration. Injections of 10 nl or more of MOs resulted in 80%
mortality, with embryos exhibiting non-specific necrosis; injections of 5-10
nl resulted in a lower mortality with embryos living through the segmentation
stages, but they still displayed high levels of necrosis. For all MOs
(foxd3, sox10, kal1.1, kal1.2) 2-4 nl produced specific defects,
including those previously reported for sox10
(Dutton et al., 2001a
), and
little general necrosis, and were therefore the volumes injected in the
experiments reported here. For all mm MOs, we injected 0.5-1 nl, which
produced no observable phenotype; volumes above 2 nl resulted in 80% mortality
due to non-specific necrosis. For injections of foxd3+sox10,
equal amounts of MOs to foxd3 and sox10 were pre-mixed.
Injection of 10 nl or greater of the combined MOs resulted in 80% mortality,
as was seen with the individual MOs. Injections were done into one- to
two-cell stage embryos (Kimmel et al.,
1995
). Injection pipettes were pulled using thin walled
borosilicate glass tubing with microfilament (OD=1.2 mm, ID=0.94 mm) on a
Sutter Puller P-2000 (Sutter Instruments).
In situ hybridization
Zebrafish embryos were staged as described in Kimmel et al.
(Kimmel et al., 1995). Embryos
were fixed in phosphate-buffered 4% paraformaldehyde. Whole-mount in situ
hybridization was performed as described in Thisse et al.
(Thisse et al., 1993
), using
single-stranded RNA probes labeled with digoxigenin-UTP (Roche), the only
modification being that the pronase (Sigma) permeabilization step was
shortened to 3 minutes. The GnRH probes were generated after cloning these
cDNAs from zebrafish (Gopinath et al.,
2004
). Briefly, the gnrh2 gene was initially cloned using
heterologous primers to the goldfish gnrh2 gene [GenBank Accession
number U30386 (Bogerd et al.,
1994
); forward primer, 5'-ATGGTGCACATCTGCAGGCT-3';
reverse primer, 5'-GTCATTTTCTCTTTTGGGAATC-3'] and the
gnrh3 gene, expressed in the terminal nerve, was cloned using primers
to the zebrafish gnrh3 sequence [GenBank Accession number AJ304429
(Torgersen et al., 2002
);
forward primer, 5'-CAGCACTGGTCATATGGTTGGCTTCCCGG-3'; reverse
primer, 5'-CACTCTTCCCCGTCTGTCGG-3']. gnrh2 and
gnrh3 cDNA was amplified by RT-PCR, the products were cloned into
pGEM-T Easy Vector System I (Promega) and the identity of the resulting clones
confirmed by sequencing (Gopinath et al.,
2004
). Probes to both gnrh2 and gnrh3 included
the sequences to the GnRH decapeptide and the GnRH associated protein (GAP) of
the pre-pro GnRHs, thereby making the probes highly specific to a given form
of GnRH. The pattern of in situ hybridization showed that the gnrh2
and gnrh3 probes had non-overlapping expression patterns, thereby
confirming this specificity (see Results).
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Immunocytochemistry
To date the molecular form of GnRH expressed in the migrating hypothalamic
cells has not been identified in zebrafish, and there is no antibody that
specifically recognizes only this group of GnRH cells (see Results). Thus, for
our analysis of hypothalamic GnRH (GnRH1), we used the LRH13 antibody
(Park and Wakabayashi, 1986),
which recognizes both the terminal nerve and hypothalamic GnRH cell
populations. Immunocytochemistry was carried out as previously described
(Whitlock et al., 2003
),
except that the embryos were fixed in 4% paraformaldehyde
(Westerfield, 1993
) with 7%
saturated picric acid for two hours at room temperature.
Double labeling for sox10-GFP and gnrh2
Embryos positive for sox10-GFP were fixed at 26-28 hpf and
protocol 5 of Schulte-Merker
(Schulte-Merker, 2002) for
immunocytochemistry/in situ hybridization double labeling was followed. The
anti-GFP antibody (Molecular Probes, rabbit polyclonal) was used at a dilution
of 1:1000 and the in situ probe for gnrh2 was that described
above.
Statistical analyses
The number of cells expressing gnrh2 (midbrain) and gnrh3
(terminal nerve) was counted in whole-mount embryos. Cell counts obtained in
different groups of fish were compared using the Wilcoxon Rank Sum Test,
comparing the medians of non-normal distributions.
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Results |
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Role of foxd3 and sox10 in the development of midbrain GnRH (gnrh2)
Prior to the migration of neural crest, the cranial neural crest
(Fig. 1C, purple) and the
region giving rise to the olfactory placodes
(Fig. 1C, red) share a common
border. We have shown previously, through lineage-tracing techniques, that the
neuromodulatory GnRH cells of the terminal nerve
(Fig. 1E) come from cranial
neural crest (Whitlock et al.,
2003) (Fig. 1A-C,
asterisks), and that the gnrh3 gene is expressed in these cells
starting at 24-26 hpf (Gopinath et al.,
2004
). Here, we investigated whether the neuromodulatory
gnrh2 cells of the midbrain (Fig.
1E), like the gnrh3 cells, also arise from neural
crest.
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Specificity of MO-induced defects
To determine whether the reductions in gnrh3 and gnrh2
cell number caused by the injection of sox10 and foxd3 MOs
were due to non-specific effects, we examined the expression of otx2,
fgf8 and six4.1 in MO-injected embryos. The otx2 gene
normally has a border of expression at the midbrain hindbrain boundary (MHB)
(Li et al., 1994), and
fgf8 is expressed in a distinct stripe of cells at the MHB
(Scholpp et al., 2003
). The
MHB is an important signaling center in the developing neural tube, and is the
region where the midbrain GnRH2 cells are first found
(Amano et al., 2004
;
White and Fernald, 1998
). We
found no detectable alterations to the pattern of otx2 or
fgf8 expression in sox10+foxd3 morphant embryos,
the class of MO-treated embryos most likely to express non-specific defects
because of their more extreme phenotype
(Fig. 3D, Fig. 4D;
Table 1). The posterior border
of otx2 expression was unaltered in sox10+foxd3
morphant animals (Fig. 5E,
arrow) when compared with that of wild-type uninjected fish
(Fig. 5A, arrow). Likewise,
sox10+foxd3 morphants showed strong otx2 expression
in the midbrain (Fig. 5F),
similar to that observed in wild-type embryos
(Fig. 5B). Consistent with the
maintenance of the MHB, expression of fgf8 at 36 hpf was present in
the sox10+foxd3 morphant animals
(Fig. 5G, arrows) in the same
pattern as was observed in wild-type uninjected fish
(Fig. 5C, arrows). Thus, both
otx2 and fgf8 expression patterns are maintained at the
midbrain-hindbrain boundary in the sox10+foxd3
morphants.
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Expression of sox10-GFP in the gnrh2 cells of the midbrain
The sox10 gene is initially expressed in pre-migratory neural
crest (12 hpf; Fig. 1A). As the
neural crest starts to migrate sox10 expression is downregulated. At
18-20 hpf, there is a small cluster of cells lying between the neural tube and
the backside of the developing optic cup
(Fig. 6A, arrows). By 24 hpf,
this gene expression is no longer apparent by in situ hybridization. Using the
sox10-GFP embryos, we were able to visualize this cluster of cells
until 28 hpf because of the perdurance of the GFP protein
(Fig. 6B, arrows, anti-GFP
antibody). Because gnrh2 is first expressed at 24-26 hpf
(Fig. 6C, arrows), as the
sox10-GFP expression was waning, we were able to double label for
gnrh2 (Fig. 6C,
arrows) and the GFP protein (Fig.
6B, arrows). A subset of the gnrh2-expressing cells lying
at the edge of the neural tube against the eye
(Fig. 6D, purple, arrowhead;
see also Fig. 6C) were also
positive for sox10-GFP immunoreactivity
(Fig. 6D, brown and purple,
arrow; see also Fig. 6B),
indicating that these cells still expressed sox10-GFP. Not all
gnrh2 cells were positive for GFP
(Fig. 6D, arrowhead),
presumably because some of the cells had already lost their sox10-GFP
signal. On average there are 5.33±0.58 gnrh2-positive cells at
24 hpf (Gopinath et al.,
2004). In our analysis of ten preparations, 2.9±0.69
(±s.e.m.) gnrh2-positive cells also contained GFP. Thus,
around 54% of the gnrh2 cells were double labeled at 24-26 hpf.
The expression of sox10 in the first two days of development is
very dynamic. At 36 hpf, sox10 expression is initiated in the region
of the terminal nerve and at 48 hpf is in the cells surrounding the
gnrh3 expression in the terminal nerve
(Fig. 7A,B). In general, it
appears that the gnrh3 (Fig.
7B, arrowhead, red) and sox10
(Fig. 7B, arrow, blue) signals
do not overlap, although some preparations showed cells that appeared to
co-express gnrh3 and sox10
(Fig. 7B, arrow with asterisk).
Within the midbrain there was no expression of sox10 at 36 hpf, and
at 48 hpf there were sox10-expressing cells in the CNS
(Fig. 7C,D, arrows). This
sox10 expression in the midbrain
(Fig. 7C,D, arrows, blue) did
not co-localize with gnrh2 expression
(Fig. 7C,D, arrowheads, red),
and most likely represents oligodendrocyte precursors
(Woodruff et al., 2001).
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|
Role of foxd3 and sox10 in the development of hypothalamic GnRH cells
Previously, we have shown that the endocrine GnRH cells populating the
hypothalamus arise from the anterior pituitary placode, and that these cells
are lost in mutants lacking the gli1 and gli2 signaling
molecules, that disrupt anterior pituitary development
(Whitlock et al., 2003). In
order to determine whether the development of the endocrine GnRH cells of the
hypothalamus was affected by knocking down foxd3 and sox10
function, we assayed for the presence of these GnRH cells in
sox10+foxd3 morphant embryos. In zebrafish, the anti-GnRH
antibody LRH13 labels both the terminal nerve and the hypothalamic GnRH cells
(Fig. 9E, arrowheads), whereas
in situ probes to gnrh3 only label the terminal nerve cells
(Fig. 9A). This suggests that
hypothalamic cells express a different form of GnRH, as occurs in fish as well
as some mammals (Lethimonier et al.,
2004
; Montaner et al.,
2002
; Somoza et al.,
2002
). To date, the gene encoding the form of GnRH expressed in
this migratory population of GnRH cells has not been identified in zebrafish.
Therefore, we used an antibody recognizing GnRH
(Park and Wakabayashi, 1986
)
to visualize the hypothalamic GnRH cells in sox10+foxd3
morphant embryos. Previously, we have shown that GnRH-IR is not expressed in
hypothalamic GnRH cells until 52-54 hpf. As shown in
Fig. 9F, hypothalamic GnRH
cells were present in sox10+foxd3 morphant embryos when
scored at 56 hpf. In spite of the altered morphology of the head, the
hypothalamic GnRH cells were found in a group
(Fig. 9F, arrows) near the
post-optic commissure (Fig. 9F,
asterisk). Thus, the combined knockdown of the Foxd3 and Sox10 proteins did
not result in the loss of the GnRH cells of the hypothalamus, suggesting that
these cells do not depend on foxd3 and sox10 gene function
for their development.
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Discussion |
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Neural crest origin of GnRH2 cells as reflected by sox10-GFP expression
In pre-migratory neural crest, the expression patterns of foxd3
and sox10 overlap extensively, but not completely
(Dutton et al., 2001b;
Odenthal and Nusslein-Volhard,
1998
). foxd3 is downregulated upon migration
(Kelsh et al., 2000
) (K.E.W.,
unpublished), and sox10 is maintained in the medial migratory route
giving rise to glia of the cranial ganglia
(Dutton et al., 2001b
). We
were able to use sox10-GFP embryos to follow the development of the
sox10 cells past 18-20 hpf, when the sox10 gene was no
longer expressed in the clusters of cells between the neural tube and eye. The
co-localization of GFP with gnrh2 at 26 hpf suggests that the
gnrh2 cells arise from this specific group of cranial neural
crest-derived cells. The expression of sox10 has been extensively
discussed as being important in the differentiation of peripheral glia and
central nervous system oligodendrocytes
(Woodruff et al., 2001
). We
have shown that sox10 is expressed in cells clustered around the
gnrh3 cells of the terminal nerve at 36 hpf, and these cells may
indeed be peripheral glia. More recently, sox10 has been shown to
play a role in neuronal differentiation in rat neural crest stem cells
(NCSCs). Constitutive expression of SOX10 allows NCSCs to maintain their
proliferative activity, thus allowing for the induction of the proneural gene
MASH1 (Kim et al., 2003
).
Here, we show that an MO-induced decrement in Sox10 expression results in a
loss of gnrh2 cells, suggesting that, like in rat NCSCs,
sox10 expression may be important for the differentiation of these
neurons. Therefore, as previously reported
(Kim et al., 2003
),
sox10 expression may allow for the maintenance of the proliferative
plasticity necessary for GnRH2 neural differentiation and, unlike in glial
subtypes, the expression is not maintained after the neurons
differentiate.
At 48 hpf, sox10 expression is maintained in the terminal nerve
and is also expressed in a cluster of cells in the midbrain
(Fig. 7C,D; Fig. 8D). The midbrain
sox10 expression does not co-localize with the gnrh2 cells
and may represent differentiating oligodendrocytes previously described in the
developing spinal cord of the zebrafish
(Park and Appel, 2003;
Park et al., 2002
). In
zebrafish, the co-expression of sox10 and olig2 within the
spinal cord is not evident until 48 hpf
(Park et al., 2002
), a time
well after we scored our morphants (36 hpf). Thus, in our experiments we would
not have been able to observe any effects on oligodendrocyte
differentiation.
Signaling pathways involved in the development of GnRH cells
The effects of reducing Foxd3 and/or Sox10 on the development of the
midbrain and terminal nerve GnRH cells suggest a neural crest origin for both
cell types. However, the relative contributions of these gene products to the
development of the two populations differed: we observed, on average, a
complete loss of gnrh2 cells in the double morphant, whereas the
gnrh3 cell population was less affected, suggesting that other genes
may be involved. This is consistent with previous work showing that the
signals patterning cranial neural crest differ, such that the crest cells
contributing to the frontal mass express different genes than those
contributing to the branchial arches (for a review, see
Santagati and Filippo, 2003).
The roles of specific transcription factors, such as sox10 and
foxd3, in neural crest development are complex. For example, in
Xenopus, sox10 is required for early development of the neural crest,
and its suppression results in the loss of foxd3 and slug
expression (Honore et al.,
2003
). In zebrafish, loss of sox10 results in the
elimination of melanocytes, as well as in the disruption of enteric ganglia
(Dutton et al., 2001a
;
Dutton et al., 2001b
). The
foxd3 gene appears to act as a transcriptional repressor in
Xenopus, where overexpression can lead to the loss of neural crest
(Pohl and Knochel, 2001
), and,
in chick, it appears to repress melanogenesis
(Kos et al., 2001
). Both
foxd3 and sox10 are necessary for the genesis of glia and
neurons in the peripheral nervous system of animals ranging from fish to
mammals (Britsch et al., 2001
;
Dutton et al., 2001b
;
Honore et al., 2003
;
Southard-Smith et al., 1998
).
The knockdown of foxd3 and sox10 most likely disrupts both
the initial genesis of neural crest, as well as the development of neuronal
and glial lineages. Future analysis of the role of other transcription factors
important for neural crest development, such as AP2
(Hilger-Eversheim et al.,
2000
; Mitchell et al.,
1991
), will allow us to further define the signaling pathways
involved in segregating the GnRH2 and GnRH3 neuronal populations in zebrafish.
Whether the neural crest origins for the GnRH2 and GnRH3 cells, and the
underlying genetic pathways reported here for zebrafish, apply to all
vertebrates will require further investigations.
Disruption of midbrain-hindbrain patterning: interpretations of the possible phenotypes
The hypothesis that GnRH2 cells originated from the neural tube was based
on the location of these cells in developing and adult animals. In the
flounder and the zebrafish, GnRH2 cells are found in a region of the midbrain
(midbrain tegmentum) in juvenile and adult animals
(Amano et al., 2004;
Gopinath et al., 2004
). In
addition, ablation studies support the idea that the GnRH2 cells have an
origin independent from that of the GnRH1 and GnRH3 cells
(Northcutt and Muske, 1994
).
It was subsequently proposed that this population arises from the germinal
zone around the third ventricle in the mesencephalic region of the neural tube
(Amano et al., 2004
;
Parhar et al., 1998
;
White and Fernald, 1998
). The
posterior border of the midbrain and regions immediately adjacent to it are
specified by fgf8, which is expressed in the midbrain-hindbrain
boundary (MHB). Establishment of midbrain and hindbrain progenitors is
independent of fgf8, but the maintenance of midbrain gene expression
appears to be dependent upon fgf8
(Jaszai et al., 2003
;
Reifers et al., 1998
). We used
the zebrafish ace mutant, which lacks fgf8 function, to
disrupt patterning of the neural tube. Our data show that the loss of
fgf8 does not result in a measurable change in the number of
gnrh2 cells, suggesting that gnrh2 cells either do not arise
from the neural tube, or arise from a region that is not dependent upon
fgf8 for patterning information.
Neural crest migration into the neural tube
The developing embryo is a study in cell migration, and the neural tube is
notable for the migration of the neural crest from its dorsal surface. In
addition, there have been reports of ventrally emigrating neural tube cells
(VENT) that leave the neural tube and contribute neurons to the peripheral
nervous system, as well as neurons and glia to the enteric nervous system
(Dickinson et al., 2004).
Furthermore, cells associated with the brain vasculature (vascular smooth
muscle cells) migrate into the central nervous system and may arise from
neural crest-derived head mesenchyme re-entering the developing brain
(Korn et al., 2002
). Thus,
there may be precedents for neural crest-derived non-neuronal cell types
re-entering the neural tube during development.
Our results indicate that the gnrh2-expressing cells of the
midbrain arise from cells whose site of origin and early molecular
characteristics are similar to those of neural crest cells. This is not the
first report of centrally located neurons arising from neural crest and
migrating into the neural tube. For example, the neurons of the mesencephalic
nucleus of the trigeminal (Mes5) have been reported to arise from neural crest
(Narayanan and Narayanan,
1978) and to then migrate to their adult location in the
mesencephalon. However, we first see gnrh2-expressing cells at 24 hpf
lying laterally within the neural tube, and, by 56 hpf, they are in two
clusters lying on either side of the midline
(Gopinath et al., 2004
). Thus,
they do not appear to be `unique primitive population along the dorsal
midline', as has been described for Mes5 neurons
(Sanchez et al., 2002
).
Furthermore, loss of fgf8 in the isthmus did not affect the number of
gnrh2 cells, as it does for the Mes5 neurons
(Hunter et al., 2001
). Our
data support the idea that gnrh2 cells arise from neural
crest-derived cells that migrate into the neural tube during development.
Neural crest origin for GnRH3 cells of the terminal nerve
In zebrafish, the terminal nerve is an easily identifiable telencephalic
population of GnRH cells in the forebrain that expresses gnrh3 early
in development (Gopinath et al.,
2004; Whitlock et al.,
2003
). In medaka, the differences in timing between the onset of
expression of gnrh3 in the terminal nerve and gnrh1 in the
hypothalamus led to the proposal that these populations do not share a common
origin in the olfactory placode (Dubois et
al., 2002
; Parhar et al.,
1998
). Consistent with this hypothesis, we have found that, in
zebrafish, GnRH3 cells of the terminal nerve have their origin in the cranial
neural crest (Whitlock, 2004a
;
Whitlock, 2004b
;
Whitlock et al., 2003
).
Whether the terminal nerve arises from neural crest or the olfactory placode
has been the subject of debate for the last century
(Von Bartheld and Baker, 2004
;
Whitlock, 2004b
). The data
presented here support and extend our previous findings that GnRH3 cells of
the terminal nerve arise from cranial neural crest, by defining foxd3
and sox10 function as being necessary for the differentiation of the
gnrh3 cells of the terminal nerve.
Endocrine GnRH1 is not affected when neural crest is disrupted
Originally, it was proposed that the endocrine and neuromodulatory GnRH
cells of the forebrain of mammals arose from the olfactory placode and
migrated to their adult location using routes along the developing olfactory
and vomeronasal nerves (Schwanzel-Fukuda
and Pfaff, 1989; Wray et al.,
1989a
; Wray et al.,
1989b
). Subsequently it was suggested, in both fish and mammals,
that there are multiple origins for these populations of GnRH cells
(Amano et al., 2004
;
Dubois et al., 2002
;
Parhar et al., 1998
;
Quanbeck et al., 1997
). Our
results further define the different developmental mechanisms underlying the
differentiation of the neuromodulatory GnRH cells and the endocrine GnRH
cells. Strikingly, we have shown that the knockdown of the kal1.1
gene resulted in a loss of the endocrine GnRH1 cells, suggesting a crucial
role for one isoform of the kallmann gene in the differentiation of
the endocrine GnRH1 cells.
The idea of a neural crest origin for GnRH2 cells is in agreement with
previous reports of GnRH2 in the neural crest-derived sympathetic ganglia in
amphibians (Troskie et al.,
1997). gnrh2 expression has also been reported in
non-neuronal, mesodermally derived structures such as bone marrow, kidney and
reproductive tissues (for a review, see
Millar, 2003
), clearly
indicating multiple origins for gnrh2-expressing cells beyond what we
have described here. Recently, it was demonstrated through an analysis of the
sequences of reported structural variants of GnRH that the midbrain
gnrh2 form is more closely related to the form found in the terminal
nerve of fishes (gnrh3) than it is to the hypothalamic forms
(gnrh1) (Lethimonier et al.,
2004
). Thus, the neuromodulatory midbrain GnRH2- and terminal
nerve GnRH3-containing cells have molecularly similar forms of GnRH (when
compared with the endocrine hypothalamic form, GnRH1), and our data support a
common embryonic origin for these populations in the cranial neural crest.
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ACKNOWLEDGMENTS |
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