1 Developmental Biology, Institute Biology 1, University of Freiburg,
Hauptstrasse 1, D-79104 Freiburg, Germany
2 GSF, Institute for Mammalian Genetics, Ingolstadter Landstrasse 1, D-85764
Neuherberg, Germany
Author for correspondence (e-mail:
driever{at}biologie.uni-freiburg.de)
Accepted 20 August 2003
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SUMMARY |
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Key words: Norepinephrine, Catecholaminergic system, Zebrafish, AP-2, Hindbrain, Mont blanc, Lockjaw, Retinoic acid, Neuronal differentiation
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Introduction |
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Several factors that control development of NA centers have been
identified. Two closely related homeodomain proteins, Phox2a and Phox2b, have
been shown to be crucial for the development of the LC
(Pattyn et al., 1999;
Pattyn et al., 2000b
). In
contrast to Phox2a, which controls development of NA neurons in the LC, Phox2b
is required for differentiation of all NA centers in the brain
(Pattyn et al., 2000b
). Phox2a
and Phox2b are also directly involved in the development of autonomic neurons
and hindbrain motoneurons (Valarche et
al., 1993
; Tiveron et al.,
1996
; Pattyn et al.,
1997
; Pattyn et al.,
2000a
). The proneural protein Mash1 is required for most NA
neurons (Hirsch et al., 1998
),
and has been shown to act upstream of the Phox2 genes
(Hirsch et al., 1998
;
Pattyn et al., 2000b
).
Recently, another homeodomain-containing protein, Tlx3, has been shown to
control the development of nearly all NA neurons in the hindbrain
independently of Phox2b (Qian et al.,
2001
). The transcriptional activator Tfap2a
(Williams et al., 1988
;
Mitchell et al., 1991
) is
expressed in hindbrain NA neurons. Tfap2a has been shown to bind to the
th and dbh promoters, and in cell culture assays may
activate expression of the key enzymes for development of the NA
neurotransmitter phenotype, TH and DBH
(Kim et al., 2001
).
Inactivation of the Tcfap2a gene in mouse revealed a critical
function of Tfap2a for development of the neural tube, craniofacial structures
and eyes (Schorle et al.,
1996
; Zhang et al.,
1996
; Nottoli et al.,
1998
). The defects in neural tube closure in Tcfap2a
mutant mice prevented adequate assessment of the Tcfap2a mutant
phenotype during neuronal differentiation. Thus, a potential role of Tfap2a
during NA differentiation in vivo has not been previously investigated.
We show that mutations isolated as mont blanc alleles
[mob (Neuhauss et al.,
1996); allelic with lockjaw
(Schilling et al., 1996
)]
affect zebrafish tfap2a. tfap2a mutant embryos develop defects in
neural crest and epibranchial placode derivatives. Analysis of the development
of the sympathetic and central nervous systems in tfap2a mutants
revealed loss of th expression in the LC, medulla and area postrema,
and sympathetic ganglia. Thus, neurons in the hindbrain and peripheral nervous
system do not express the noradrenergic transmitter phenotype in the absence
of tfap2a function. Furthermore, we show that retinoic acid (RA) is a
necessary inducer of NA cell fate in the hindbrain in vivo. neckless
mutant embryos, which have a disrupted raldh2 gene and are thus
deficient in RA synthesis (Begemann et al.,
2001
), lack NA neurons in the medulla oblongata. Moreover,
exogenously applied retinoic acid can upregulate tfap2a expression
and induce ectopic NA neurons in both the hindbrain medulla and the
sympathetic nervous system. By contrast, in tfap2a mutant embryos RA
fails to induce ectopic th expression in the hindbrain or sympathetic
nervous system. Taken together, our results provide evidence that the
neurotransmitter phenotype of hindbrain NA neurons is regulated by
tfap2a. Our data further reveal distinctive control mechanisms for NA
neurons in the LC and medulla: Although LC neurons are specified through a
pathway including Fgf8 and phox2a
(Guo et al., 1999a
), neurons
of the medulla are induced by RA, which we have identified as acting upstream
of tfap2a.
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Materials and methods |
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To simplify cross-reference with the mammalian literature, we will use the approved UCL/HGNC/HUGO Human Gene Nomenclature database symbol tfap2a for the zebrafish gene, instead of a gene name derived from the allele designations of the initial isolates mont blanc mobm610/780, mobm819 and lockjaw (lowts213). The use of the gene name tfap2a for the zebrafish gene will also avoid confusion with other genes that have been named AP2 (e.g. adaptor protein 2).
Mapping and cloning of mob/tfap2a
We mapped mobm819 using bulked segregant analysis
(Michelmore et al., 1991) with
pooled DNA from homozygous mutants and siblings from a
mobm819 AB x India map-cross. We linked
mobm819 to linkage group 24 between SSLP markers Z23011
and Z13695. Linkage with tfap2a was determined by RFLP analysis of
PCR products from DNA of single mobm819 mutant and
wild-type sibling embryos, obtained using primers flanking exon 5.
DraII restriction digest of the amplified tfap2a products
did not digest the 360 bp PCR product from wild-type embryos, but cleaved that
of mobm819 mutant embryos into two fragments of 123bp and
237bp. Analysis of the mobm610 and
mobmm780 alleles reported previously
(Neuhauss et al., 1996
)
revealed that they are likely re-isolates of one allele, as both represent the
same mutational event: a point mutation in the splice acceptor at the 5'
end of exon 7 resulting in the use of a cryptic splice acceptor 14 bp further
3', which causes a frame shift in the DNA-binding and dimerization
domain, and a truncation at amino acid 395 (A.B. and E.K., unpublished).
Sequencing the EST fc31a07 revealed that it contained the full-length ORF of tfap2a isoform 3. Using gene specific primers for this EST, we performed RT-PCR with mRNA from mobm819 mutant and wild-type embryos to obtain the full-length ORF. Total RNA was isolated using the RNAeasy kit (Qiagen) and RT-PCR was performed with the RevertAid cDNA synthesis kit (MBI Fermentas). PCR products were cloned into pCR4 vector using the TOPO TA kit (Invitrogen). Sequencing was carried out on an automated sequencer (MWG). The full-length sequence of tfap2a isoform 3 has been submitted to GenBank (Accession Number AY166856).
Retinoic acid and cycloheximide treatment
Embryos from wild-type or heterozygous mobm819 parents
were exposed to serial dilutions (3x108 to
3x106 M in egg water) of all-trans retinoic acid (RA,
Sigma; stock solution 3x101M in DMSO) for defined time
periods in the dark. After treatment, embryos were washed under safety red
light three times each for 10 minutes with egg water
(Westerfield, 1995) to remove
RA, and fixed at desired stages. For th expression analysis zebrafish
embryos were exposed to RA for 9 hours between 24 hpf and 33 hpf, or for 24
hours between 24 hpf and 48 hpf. Hox gene expression analysis was performed on
zebrafish embryos treated with RA for 9 hours between 24 hpf and 33 hpf.
Cycloheximide (CHX, Calbiochem; stock of 10 mg/ml in 100% ethanol) was applied at a concentration of 100 µg/ml in egg water. Embryos were exposed to CHX from 21 hpf to 32 hpf. For combined CHX and RA treatments, RA was added at 24 hpf to the embryos (final concentration of 3x107 M RA) and incubated for 8 hours. At 32 hpf, the embryos were washed as described above and fixed at desired stages. Controls for each experiment included sibling embryos incubated in the same concentration of Ethanol and/or DMSO as the experimental embryos.
Whole-mount in situ hybridization and immunohistochemistry
Whole-mount in situ hybridization (WISH) was performed according to
Hauptmann and Gerster (Hauptmann and
Gerster, 1994). Digoxigenin- or fluorescein-labeled antisense RNA
probes were prepared using RNA labeling reagents (Boehringer).We prepared RNA
probes for the following genes: th
(Holzschuh et al., 2001
);
phox2a (Guo et al.,
1999a
); hoxa2, hoxb2, hoxb3 and hoxd3
(Prince et al., 1998
);
isl1 (Inoue et al.,
1994b
); and ret1
(Bisgrove et al., 1997
). To
generate a probe specific for dopamine beta hydroxylase
(dbh) expression, we used a PCR-based approach to combine genomic
fragments (Sanger Centre, Hinxton, UK) which contain Dbh-coding region, as
judged from the sequence two genomic fragments (z35723-a1914e04.q1c and
zfishC-a1746c09.p1c) in public databases and from comparison with mammalian
Dbh sequences. To generate the probe containing 735 bp of the dbh
coding sequence, we linearized with SpeI and transcribed with T7
polymerase. To make tfap2a probes, the EST fc31a07 was linearized
with SalI (MBI Fermentas) and RNA probe was transcribed with Sp6 RNA
polymerase (Boehringer). Immunohistochemistry anti-serotonin antibodies
(Chemicon), anti HuC/HuD antibodies (16A11, Molecular Probes), and
anti-acetylated tubulin antibodies (Sigma) was performed as described
(Solnica-Krezel and Driever,
1994
), except that fixed embryos older than 3 dpf were
permeabilized by 30 minutes incubation in proteinase K (Sigma; 10 µg/ml)
and successive inhibition of proteinase K with phenylmethyl-sulfonylfluoride
(PMSF, Sigma). The cartilage of zebrafish embryos was stained with Alcian Blue
as described elsewhere (Neuhauss et al.,
1996
).
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Results |
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mont blanc encodes the transcription factor Tfap2a
To identify the gene affected by mont blanc (mob)
mutations, we mapped the mutation using simple sequence length polymorphism
markers (SSLP). mob is located between the markers z23011 (2.04 cM
from mob; 11 recombinants in 538 meiosis) and z13695 (2.75 cM from
mob; 15 recombinants in 546 meiosis) on LG 24
(Fig. 2A). Two EST clones map
to this region: fc31a07 (W. Talbot,
http://zebrafish.stanford.edu)
and fb83f04 (I. Dawid,
http://dir.nichd.nih.gov/lmg/lmgdevb.htm),
both predicted to be homologous to mammalian Tfap2a (S. Johnson, AI584805,
AI722745; WUZGR;
http//zfish.wustl.edu).
Tcfap2a mutant mouse embryos
(Schorle et al., 1996;
Zhang et al., 1996
) develop
craniofacial and PNS phenotypes similar to those of
mobm819 zebrafish mutants. Thus, tfap2a appeared
to be a likely candidate gene for mont blanc mutations. As in mouse,
the zebrafish tfap2a locus encodes three mRNA isoforms, each using
different first exons. Zebrafish tfap2a isoforms tfap2a1 and
tfap2a2 have been isolated (M. Fuerthauer, C. Thisse and B. Thisse,
GenBank Accession Numbers AF457191, AF457192). We cloned and sequenced a
full-length cDNA that encodes the zebrafish tfap2a3, a homolog of the
mouse Tcfap2a isoform 3. The amino acid sequences of all three Tfap2a
isoforms are highly conserved: isoforms 1 and 3 each share 85% amino acid
identity between mouse and zebrafish, while isoform 2 shares 82% identity. The
three zebrafish isoforms differ from each other only in the first 15 (isoform
1), 21 (isoform 2) or 9 (isoform 3) amino acids, which are encoded by three
alternative first exons (Fig.
2). We assembled the genomic structure of tfap2a
(Fig. 2B) through in silico
chromosome walk on publicly available zebrafish genomic sequences
(http://www.sanger.ac.uk/Projects/D_rerio).
Sequencing of cDNAs from homozygous mobm819 embryos
identified a point mutation in exon V, close to the splice-acceptor site
(Fig. 2C). This A to T
transition created a DraII restriction site, which made it possible
to confirm linkage to mobm819 by RFLP analysis
(Fig. 2C,D). We did not find a
single recombinant in 546 meiosis, indicating that mob and
tfap2a map within an interval smaller than 0.18 cM. The mutation
introduces a stop codon that truncates the protein at amino acid 264, located
at the N-terminal end of the dimerization and DNA-binding domain (DDB;
Fig. 2C,E). This domain is
necessary for the dimerization of Tfap2 proteins, which in turn is required
for DNA binding of Tfap2a protein to the target promoters and the formation of
a functional transcriptional activation complex
(Williams and Tjian, 1991a
;
Williams and Tjian, 1991b
).
Therefore, mobm819 is most likely a null allele. All three
tfap2a isoforms are affected by the mutation.
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Expression of dlx2 and ret1 is greatly reduced in tfap2a mutant embryos at 24 hpf (Fig. 4A-D). As in wild-type embryos, at this stage the neural crest and the placodal-derived cells migrate to their final positions. Later in development, when the sensory ganglia of the VIIth (facial), IXth (glossopharyngeal) and Xth (vagal) cranial nerves are forming, expression of ret1 and phox2a is not detectable above the branchial clefts in mutant embryos (Fig. 4E-H). However, the anterior and posterior lateral line ganglia express phox2a. We further analyzed tfap2a mutant embryos by immunohistochemistry with anti-acetylated tubulin antibodies to visualize axon tracts. In tfap2a mutant embryos, the early axon scaffold tracts of the CNS appear normal at 24 hpf (data not shown). By 4 dpf, however, the pattern of peripheral axons in the jaw and pharyngeal regions of mutant embryos is disturbed, suggesting that either the fibers of the VII and IX cranial nerves are absent, or that they fail to reach this region because craniofacial structures are disturbed (Fig. 4I,J).
phox2a and tfap2a expression in the locus coeruleus
and the medulla
The homeodomain transcription factor Phox2a is expressed by noradrenergic
neurons in the locus coeruleus and is required for their development in
mammals and zebrafish (Morin et al.,
1997; Pattyn et al.,
1997
; Guo et al.,
1999a
). The phox2a gene is disrupted in zebrafish
soulless (sou) mutant embryos, in which NA neurons of the LC
do not form (Guo et al.,
1999a
). Tfap2a and tyrosine hydroxylase are co-localized in
noradrenergic neurons in the embryonic mouse hindbrain
(Kim et al., 2001
). To test
whether tfap2a and phox2a are co-expressed in zebrafish
noradrenergic neurons of the hindbrain, we performed double in situ
hybridizations with tfap2a and phox2a
(Fig. 5A-C,E-J) or th
probes (Fig. 5D).
phox2a and tfap2a are indeed co-expressed in the zebrafish
LC (Fig. 5C,G,I). When the
first cells in the posterior hindbrain differentiate into NA neurons between
36 and 48 hpf, the expression domains of phox2a and tfap2a
overlap in the ventral half of the hindbrain
(Fig. 5E,F). tfap2a
expression also expands to more dorsal areas where NA neurons differentiate.
Consistent with the fact that phox2a is not expressed in the part of
the medulla/area postrema where NA neurons develop, we find normal NA
differentiation in the medulla of soulless/phox2a mutant embryos
(Fig. 5M,N).
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Sympathetic neurons fail to differentiate in tfap2a
mutants
Dorsal root ganglia (DRG), enteric and sympathetic ganglia are neuronal
derivatives of trunk neural crest. The absence of th and dbh
expression from the intestinal tract of mob/tfap2a mutants could
either reflect a differentiation defect resulting in absence of terminal
differentiation markers, or could be caused by the mutation affecting neural
crest precursor cells, and thus resulting in migration defects or reduced
survival. To test whether precursors of trunk neural crest-derived peripheral
neurons initially form, we analyzed Hu-antigen and isl1 expression.
The anti-Hu antibody recognizes a subset of ELAV-related proteins expressed
very early in neuron differentiation
(Marusich et al., 1994a). At
72 hpf the presence of Hu-immunoreactive cells reveals that precursors of
sympathetic ganglia as well as enteric neurons of the anterior intestine are
present at similar cell numbers in wild-type and mob/tfap2a mutant
embryos (Fig. 6A,B). In
contrast, we did not detect enteric neurons colonizing the posterior part of
the gut tube in mob/tfap2a mutant embryos
(Fig. 6C,D). DRG and
Rohon-Beard cells (RB; dorsal primary sensory neurons of lateral neural plate
origin in close lineage relation with neural crest)
(Artinger et al., 1999
) develop
in mob/tfap2a mutant embryos, thought at 48 hpf they appear slightly
reduced in cell number (Fig.
6E,F; for further discussion of DRGs in mob see A.B.-G.
and E.W.K., unpublished). The LIM homedomain containing transcription factor
Isl1 is expressed during early differentiation of dorsal root and sympathetic
ganglia, as well as other neurons (Ericson
et al., 1992
; Inoue et al.,
1994a
). In 72 hpf old mob/tfap2a mutant embryos cells
express isl1 at the location where sympathetic neurons form in wild
type (Fig. 6G,H). Furthermore,
phox2a is expressed in mob/tfap2a mutant embryos in cells at
the location where sympathetic neurons form in wild type
(Fig. 6I,J). Taken together our
data suggest that the lack of th and dbh expression for the
noradrenergic transmitter phenotype is not caused by a failure of neural crest
formation or migration, but reflects a block in terminal differentiation of
sympathetic neurons.
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Induction of th expression by RA signaling may be mediated by
Tfap2a
RA signaling is mediated by two classes of retinoid receptors, RARs and
RXRs (Chambon, 1995). To
determine whether RA may directly regulate the expression of th
through its receptors, or whether it may act through the expression of
intermediary proteins, we tested whether RA can induce ectopic th
expression in the absence of new protein synthesis. To inhibit protein
synthesis, we exposed zebrafish embryos to the protein synthesis inhibitor
cycloheximide shortly before and during RA treatment. Embryos treated with
cycloheximide alone had normal th expression in the CNS
(Fig. 9B,F,J), but only a few
th-expressing sympathetic neurons formed
(Fig. 9N). Embryos treated with
cycloheximide and RA did not express th ectopically in the hindbrain
or peripheral nervous system (Fig. 9
compare C,G,K,O with D,H,L,P). Thus, protein synthesis is required
to mediate the effect of RA on NA differentiation, indicating the involvement
of intermediate targets of RA. To investigate whether tfap2a may
mediate RA induced ectopic expression of th in the hindbrain, we
analyzed tfap2a expression in RA-treated embryos. After RA exposure,
tfap2a expression was upregulated in a restricted region of the
hindbrain, in which th is also ectopically expressed in response to
RA treatment (Fig. 9Q,R). We
also examined the expression of phox2a and tfap2a in
nls mutant embryos to elucidate if a reduction of RA signaling
affects the expression pattern of these genes. At 34 hpf, tfap2a and
phox2a expression was reduced in the hindbrain posterior to
rhombomere 5 (Fig. 9S,T). These
findings correlate with the lack of th-expressing cells in the
posterior hindbrain and the normal development of the LC in nls
mutants.
The failure of RA to induce ectopic th expression in the hindbrain when protein synthesis was blocked indicates that the response to RA treatment requires intermediary factors. The upregulation of tfap2a expression after RA treatment and the downregulation of tfap2a expression in RA-deficient nls mutant embryos suggest that Tfap2a may mediate RA induced th expression in the posterior hindbrain.
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Discussion |
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The development of neural crest and epibranchial placode derivatives
of the autonomic nervous system depend on tfap2a function
Neural crest cells arise from the dorsal neural tube, migrate throughout
the body and differentiate into many different derivatives, including
peripheral nervous system and craniofacial skeleton (reviewed by
Le Douarin and Kalcheim,
1999). Loss-of-function experiments in mouse revealed that
tfap2a contributes to a wide variety of developmental events,
including skeletal formation in the thorax and limbs, aspects of
organogenesis, formation of the peripheral nervous system, and neural tube
closure (Schorle et al., 1996
;
Zhang et al., 1996
). The
analysis of tfap2a chimeric mice has shown that Tfap2a is indeed
autonomously required for each of the pleiotropic aspects of the mutant
phenotype investigated to date (Nottoli et
al., 1998
). In zebrafish, mutations in mob/tfap2a also
cause extensive defects in the development of neural crest derivatives,
including the craniofacial skeleton and pigment cells
(Neuhauss et al., 1996
;
Schilling et al., 1996
).
Primary sensory neurons of the trunk arise exclusively from the neural
crest. By contrast, only some of the cranial sensory neurons are derived from
neural crest. The majority of cranial sensory neurons have their origin in the
dorsolateral trigeminal and vestibular placodes, and the distal epibranchial
placodes (Ayer-Le Lievre and Le Douarin,
1982; D'Amico-Martel and
Noden, 1983
; Noden,
1993
). The loss of the epibranchial derived sensory ganglia in
tfap2a mutant embryos could be explained by defects in neural crest
derived cranial structures. It was shown that neurogenic placodes form under
the influence of the surrounding cranial tissues, including neural crest and
pharyngeal endoderm (Vogel and Davies,
1993
; Begbie et al.,
1999
). The reduction of phox2a and ret1
expression in tfap2a mutants indicates that both components of the
sensory ganglia, namely neural crest and neurogenic placodal cells, are
directly affected by lack of tfap2a. We have not detected an increase
in apoptosis in any of the cranial ganglia in mob/tfap2a mutants
(data not shown). This is in contrast to Tcfap2a knockout mice, which
develop increased apoptosis in the trigeminal ganglia
(Zhang et al., 1996
).
tfap2a is expressed in neural crest cells and required for their
normal development (Luo et al.,
2003), and thus it is likely that neural crest defects in
tfap2a mutants mediate the sensory ganglia phenotype. By contrast,
the phenotype in the sympathetic nervous system may reflect a direct
involvement of tfap2a in specification of the NA neurotransmitter
phenotype. Hu and isl1 expression occurs early in the maturation
process and is observed in S-phase neural precursor cells of the sympathetic
ganglia (Vogel et al., 1993
;
Marusich et al., 1994b
;
Avivi and Goldstein, 1999
). The
expression of Hu and isl1 in cells in the sympathetic ganglia of
tfap2a mutants indicates that neural crest cells migrate to their
proper location, proliferate and start to differentiate, but fail to adopt
their neurotransmitter phenotype. Thus, it is likely that expression of
tfap2a in sympathetic precursors is required for correct further
development and differentiation towards the noradrenergic phenotype.
tfap2a is required for noradrenergic identity of brainstem
neurons
Two main groups of noradrenergic (NA) neurons, a caudal rhombencephalic
group (A1-A3, in medulla and area postrema) and a rostral rhombencephalic
group (A4-A7, in the locus coeruleus), can be recognized in the hindbrain of
higher vertebrates (Smeets and Reiner,
1994; Smeets and Gonzalez,
2000
) as well as in zebrafish
(Ma, 1994a
;
Ma, 1994b
;
Ma, 1997
;
Holzschuh et al., 2001
). The
control of noradrenergic development in the mouse hindbrain has mainly been
studied in the locus coeruleus (reviewed by
Goridis and Rohrer, 2002
),
where important roles have been demonstrated for Mash1
(Hirsch et al., 1998
), Phox2a
(Morin et al., 1997
), Phox2b
(Pattyn et al., 1999
) and Tlx3
(Qian et al., 2001
). Although
Mash1, Phox2a and Phox2b appear to act in a linear cascade
(Hirsch et al., 1998
;
Pattyn et al., 2000a
), Tlx3 is
thought to act independently of Phox2b during LC development
(Qian et al., 2001
).
Tcfap2a is expressed in NA and AD neurons in the mouse and has been
implicated to contribute to activation of the TH and DBH promoters in cell
culture systems (Kim et al.,
1998
; Kim et al.,
2001
); however, a function of Tcfap2a for NA
differentiation in vivo was unknown. We demonstrate a novel requirement of
Tfap2a for NA neuron differentiation in the LC.
The development of the NA groups in the posterior hindbrain has not been
studied extensively. It was shown that Tlx3 and Phox2b are required for the
development of medullary NA neurons
(Pattyn et al., 2000a;
Qian et al., 2001
). We show
that expression of th is missing in the entire hindbrain of
tfap2a mutant embryos at 2 dpf when noradrenergic differentiation
occurs in wild type. Thus, our studies reveal tfap2a as an additional
transcription factor required for early NA neuronal differentiation throughout
the hindbrain. However, low level expression of dbh can be detected
in a small group of cells in the medulla/area postrema from 4 dpf onwards,
which indicates that some NA neuronal precursors can bypass a requirement for
tfap2a during late development. We can only speculate that other
transcription factors with later onset of expression, possibly additional
members of the Tfap2 family, may substitute for tfap2a.
A loss of NA identity of the LC has also been reported for zebrafish
soulless/phox2a mutant embryos, in which LC progenitor cells have
been shown to exist (Guo et al.,
1999a). We find that the medullary and area postrema NA neurons
develop normally in soulless/phox2a mutants, in contrast to
tfap2a mutant embryos. The genetic requirement for both
tfap2a and phox2a to control th expression in the
LC could be explained either by both of them providing independent inputs for
NA specification, or by tfap2a acting downstream of phox2a
in specification of the NA neurotransmitter phenotype. The latter idea would
be consistent with our findings that the loss of functional Tfap2a does not
cause a loss of phox2a expression in the LC, while th
expression does not occurs in tfap2a mutant embryos. Thus,
tfap2a could be a downstream target of soulless/phox2a.
However, several observations argue against this hypothesis being true for the
whole hindbrain. First, tfap2a expression is not detected in many
phox2a-expressing cells in the hindbrain and vice versa. Second, the
expression pattern of tfap2a is not altered in
soulless/phox2a mutant embryos. Third, phox2a and
tfap2a are not co-expressed in cells that give rise to NA neurons of
the medulla/area postrema. Therefore, tfap2a and phox2a may
provide parallel inputs into NA differentiation of LC neurons, while upstream
factors other than phox2a, e.g. phox2b, may control
tfap2a expression in the medulla/area postrema.
The role of retinoic acid in NA neuron specification
The capacity of RA to induce neuronal differentiation in cell culture has
been well documented for several cell lines (reviewed by
Guan et al., 2001).
Furthermore, the expression of Tfap2a is enhanced during RA induced neuronal
differentiation in a human teratocarcinoma cell line
(Williams et al., 1988
;
Luscher et al., 1989
). In cell
culture, RA may induce the expression of the noradrenaline transporter, a
protein specifically expressed in NA neurons
(Matsuoka et al., 1997
). RA
may also increase the number of TH-positive cells in quail neural crest
culture (Rockwood and Maxwell,
1996
). These findings suggest that the RA signaling pathway may
play a role in NA differentiation. To investigate a potential role of RA
during zebrafish NA development in vivo, we analyzed embryos that are
genetically deficient in one of the RA synthesis pathway enzymes,
retinaldehyde dehydrogenase 2 (Raldh2), as well as embryos that have been
exposed to high levels of RA. Incubation of embryos in the presence of high RA
concentrations induced ectopic th expression in the anterior
hindbrain caudal to rhombomere 1, as well as in the sympathetic nervous
system. The capacity of RA to induce ectopic th-expressing cells was
clearly restricted to rhombomere 2 to 7, and never occurred in rhombomere 1 or
in the proximity of the LC. The nuclear transducers of RA signaling are the
RAR- and RXR-type retinoid receptors, which bind to retinoic acid response
elements (RARE) in target gene promoters as RXR homodimers, or as RXR
heterodimers either with RAR, or with other nuclear receptors
(Gudas, 1994
;
Chambon, 1995
). Several
zebrafish retinoic acid receptor genes have been cloned
(Joore et al., 1994
;
White et al., 1994
;
Jones et al., 1995
). At 24
hpf, rara is expressed in the hindbrain with a sharp posterior border
at rhombomere 6 to 7. rarg is also expressed in the ventral part of
the hindbrain and in two lateral patches of head mesenchyme
(Joore et al., 1994
).
Following RA treatment, the expression of rarg is induced in the
entire brain, whereas rara is ubiquitously induced in the embryo
(Joore et al., 1994
). As
rara and rarg are both expressed in the posterior hindbrain
and induced ectopically by RA, both are potential mediators for RA induced
th expression in the hindbrain. The fact that both receptors are not
expressed in rhombomere 1 may explain why RA is unable to induce th
expression anterior to rhombomere 2. When exposing zebrafish embryos to the
protein synthesis inhibitor cycloheximide, we prevented the induction of
ectopic th expression by RA. Therefore, translation of an
intermediary target of RA signaling is required to induce th
expression, rather than th being directly activated by retinoid
receptors.
We showed that, after treatment of embryos with excess RA, tfap2a
expression is upregulated in the posterior hindbrain. Furthermore, in
tfap2a mutant embryos, exogenously applied RA is not able to
ectopically induce th expression or to rescue the deficiency in
NA-expressing cells. These findings implicate that RA signaling may induce
th expression via activation of tfap2a expression. Indeed, a
requirement for RA was confirmed when we analyzed zebrafish neckless
/raldh2 mutant embryos, which are deprived of RA. Raldh2 is involved in
the biosynthesis of RA from vitamin A (reviewed by
Duester, 2000). Loss of
function of Raldh2 in mouse mimicked the most severe phenotypes associated
with vitamin A deficiency (VAD), implicating Raldh2 as the main source of RA
in the vertebrate embryo (Niederreither et
al., 1999
; Niederreither et
al., 2000
). Zebrafish neckless/raldh2 mutant embryos
resemble many aspects of VAD (Begemann et
al., 2001
). In nls mutants, we find that the NA neurons
are missing in the posterior hindbrain, and that the expression of
tfap2a is downregulated.
It is well established that RA signaling is involved in AP patterning of
the hindbrain by regulating the expression of Hox genes
(Holder and Hill, 1991;
Hill et al., 1995
) (reviewed
by Gavalas and Krumlauf, 2000
;
Gavalas, 2002
). In our
experiments, however, embryos were exposed to RA only from 24 hpf onwards,
when rhombomere identity has already been determined in the zebrafish. Indeed,
we can demonstrate that the AP patterning of the hindbrain is not changed in
our experiments: Anterioposterior expression borders of hoxa2, hoxb2,
hoxb3, hoxd3 and epha4 were unaffected in our RA-treated
embryos. Similarly, treatment of chick embryos with an RA antagonist at late
or post-somitogenesis stages has not resulted in any changes of rhombomere
identity (Dupe and Lumsden,
2001
).
In summary, our data provide evidence that two separate mechanisms control
noradrenergic development in the zebrafish hindbrain. In the locus coeruleus,
signals from the midbrain-hindbrain-boundary organizer initiate a cascade of
transcription factors, including Phox2a and Phox2b
(Guo et al., 1999a), which
ultimately requires Tfap2a for NA precursor cells to express the NA
neurotransmitter phenotype. In the posterior hindbrain, the medulla and area
postrema, retinoic acid is an important signal, both necessary and sufficient
to induce NA differentiation, and precursor neurons share a requirement for
Tfap2a in order to be able to express the NA neurotransmitter phenotype. Thus,
although the inductive signals may be different, hindbrain NA neurons of the
LC and the posterior groups share a requirement for Phox2b, Tlx3 and Tfap2a to
establish their noradrenergic identity. Furthermore, our data reveal that
noradrenergic precursors of the peripheral nervous system rely on
tfap2a to adopt their neurotransmitter phenotype.
![]() |
ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Ramón y Cajal CSIC, Avenida Dr Arce 37, Madrid
28002, Spain
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