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First published online February 20, 2009
doi: 10.1242/10.1242/dev.033878
1 Developmental Biology, Institute Biology I, Faculty of Biology, University of
Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany.
2 Freiburg Institute for Advanced Studies, University of Freiburg, Albertstrasse
19, D-79104 Freiburg, Germany.
Author for correspondence (e-mail:
driever{at}biologie.uni-freiburg.de)
Accepted 26 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Diencephalon, Hypothalamus, Dopaminergic neurons, Neurosecretory cells, otp, arnt2, sim1, Neural differentiation
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Marín et al., 2005;
Prakash and Wurst, 2006;
Björklund and Dunnett,
2007). Mammalian diencephalic DA cell groups are classified A11
through A15 from caudal to rostral. In zebrafish (Danio rerio), DA
neurons are exclusively located in the forebrain, with the majority of groups
in the ventral diencephalon located in ventral thalamus, posterior tuberculum
and hypothalamus. However, no DA neurons can be detected in the midbrain
(Holzschuh et al., 2001;
Kaslin and Panula, 2001;
Rink and Wullimann, 2002;
McLean and Fetcho, 2004).
Despite the large evolutionary distance between teleosts and mammals, and the
differences in neuroanatomy, some anatomical and functional homologies between
zebrafish DA neuron groups and their mammalian counterparts have been
established (Smeets and Gonzalez,
2000). Only a few signaling and transcription factors involved in
zebrafish DA neuron development are known, and a detailed understanding how
specific DA neuronal groups are formed is lacking (reviewed by
Ryu et al., 2006) However,
recent data suggest that distinct DA neuron groups located in the ventral
diencephalon of zebrafish share requirement for the homeobox protein
Orthopedia (Otp) with neuroendocrine cells in the anterior hypothalamus of
mammals (Ryu et al.,
2007).
The neurosecretory system of the hypothalamus consists of two major types
of neurons: magnocellular neurons in the paraventricular nuclei (PVN) and
supraoptic nuclei (SON). These neurons synthesize oxytocin (OT) and arginine
vasopressin (VP), and project their axons to the posterior lobe of the
pituitary. Parvocellular neurons of the PVN and anterior periventricular
nuclei (APV), conversely, project to the vasculature of the median eminence
where they release their peptides, including corticotropin-releasing hormone
(CRH), thyrotropin-releasing hormone (TRH) and somatostatin (SST). Genetic
analysis in mice has provided evidence that a heterodimer of the two bHLH-PAS
transcription factors aryl-hydrocarbon receptor nuclear translocator
2 (arnt2) and single-minded 1 (sim1) is
required to specify magnocellular neurons of the PVN as well as parvocellular
CRH, TRH, SST neurons of the APV, PVN and SON
(Michaud et al., 1998;
Michaud et al., 2000;
Hosoya et al., 2001;
Keith et al., 2001). bHLH-PAS
transcription factors are important modulators of a wide range of
physiological and developmental processes, including control of aspects of
neural development (reviewed by Crews and
Fan, 1999; Gu et al.,
2000; Kewley,
2004). Members of the bHLH-PAS protein family are characterized by
an N-terminal bHLH domain required for DNA binding and dimerization, as well
as by a PAS domain that acts as a secondary dimerization interface
(Crews and Fan, 1999;
Taylor and Zhulin, 1999).
Transcriptional control of target genes requires heterodimerization of class I
and class II bHLH-PAS transcription factors
(Ema et al., 1996). The
bHLH-PAS class I factors of the Sim type exclusively form heterodimers with
class II members Arnt and Arnt2.
In addition to Arnt2 and Sim1, the Otp transcription factor is essential
for development of the same neurosecretory cell types through a parallel
pathway (Acampora et al., 2000;
Wang and Lufkin, 2000). While
Arnt2/Sim1 act during late differentiation of postmitotic progenitors, Otp is
involved both in proliferation and differentiation of neuroendocrine
progenitors. In case of AVP, CRH and OT cells, Arnt2/Sim1 and Otp act upstream
of the POU-domain protein Pou3f2/Brn2. Consistent with this, mice mutant for
pou3f2 show defects in terminal differentiation of AVP, CRH and OT
cell lineages (Schonemann et al.,
1995; Sharp and Morgan,
1996; Treier and Rosenfeld,
1996).
For lower vertebrates, including zebrafish, the contributions of Arnt2,
Sim1 and Otp to diencephalic development are less well understood. We and
others have previously demonstrated that development of a specific subset of
DA neurons in the hypothalamus and posterior tuberculum is dependent on Otp
function (Blechman et al.,
2007; Del Giacco et al.,
2006; Ryu et al.,
2007). Otp mutant mice are deficient in A11 DA neurons of
the pretectum and hypothalamus, revealing an evolutionary relationship between
these groups in mammals and teleost (Ryu
et al., 2007). Otp is essential for specification of
crh-, sst- and isotocin- (itnp; the
zebrafish ortholog of mammalian OT) expressing cells in the preoptic region of
the hypothalamus (PO) (Blechman et al.,
2007; Ryu et al.,
2007). Knockdown of zebrafish sim1 leads to reduction or
loss of itnp- and vasotocin- (vsnp; the zebrafish
ortholog of mammalian VP) expressing cells in the zebrafish preoptic region
(Eaton and Glasgow, 2007;
Eaton et al., 2008). Thus, Otp
and Sim1 appear to be involved in specification of various neuronal subtypes
in the zebrafish diencephalon.
In order to identify novel determinants for DA neuron development in the zebrafish forebrain, we screened mutations with abnormal DA systems, and isolated a zebrafish mutant line in which the bHLH-PAS transcription factor Arnt2 is disrupted. Here, we explore the function of Arnt2, Sim1 and Otp in DA neuron and neuroendocrine specification in zebrafish.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To inhibit pigmentation, embryos were incubated in 0.2 mM
phenylthiourea.
The arnt2m1055 mutant allele (TU allele number
t30256) was isolated from an ENU mutagenesis screen
(www.zf-models.org).
Other strains used are arnt2hi2639c
(Golling et al., 2002) and
otpam866 (Ryu et al.,
2007). For the Tg(hsp70otpa;hsp70YFP) line
otpa, full-length coding sequence from pCS2-otpa
(Ryu et al., 2007) was
inserted into a dual heat shock promoter expression plasmid (Dirk Meyer,
Innsbruck, unpublished) to obtain hsp70otpa;hsp70YFP. A single G0
founder (allele designation m1072) was used to create a stable F1
family. In this study, transgenic F2 fish were exclusively used. Analysis of
F3 hsp70otpa;hsp70YFP embryos showed that the otpa and
YFP cassettes segregate independently (potentially caused by
recombination over the dual hsp70 or polyA sequences in independent
transgenes). We therefore could not use YFP expression as a reporter for Otpa
overexpression. We refer to the established line as hs:otpa.
For heatshock experiments, only F3 embryos derived from incrosses of hs:otpa transgenic fish were used for which it had been demonstrated that all embryos overexpressed otpa. For verification, embryo clutches were staged and fixed at desired time points. In one half of the fixed embryos, overexpression of otpa mRNA was confirmed by whole-mount in situ hybridization, the other half was used for analysis of experimental gene expression. All heatshocks were performed by transferring embryos to 39°C preheated water for 75 minutes starting at 9 hpf. To facilitate detection of otherwise low levels of ectopic otpa mRNA in embryos fixed later than 25 hpf, a second heatshock at 39°C for 45 minutes was conducted 2-3 hours before fixation.
Mapping and cloning of arnt2m1055
Chromosomal landing of arnt2m1055 was performed using
SSLP marker (Knapik et al.,
1998; Geisler et al.,
2007). Fine mapping was carried out using DNA from single embryos.
Information on relevant SSLP markers in the critical interval was obtained
from the MGH and T51 panels. Full-length arnt2-coding sequence was
amplified from cDNA of 3 dpf arnt2m1055 mutant embryos and
wild-type siblings (forward, 5'-GGCAATATGGCAACACCAGCCGCTG-3';
reverse, 5'-TTCATGTGCGTCAGGTGTGAGATGAAGG-3') and sequenced.
arnt2m1055 embryos derived from TÜxWIK
map-crosses were genotyped using the closest SSLP marker sc1024.8_2 (forward,
5'-CTGCACGCAGAACAAATGAT-3'; reverse,
5'-CACCTGCCAAAACACTCTCA-3'). arnt2m1055
embryos in TÜ background were genotyped using a common reverse primer in
combination with mismatch forward primers whose 3' ends preferentially
hybridize with either mutant or wild-type sequences (mutant forward,
5'-TCGGCTCCAGTCACAGCTGA-3'; wild-type forward,
5'-TCGGCTCCAGTCACAGCTGT-3'; common reverse,
5'-TTGCGATTGGCGAGGTTGGAGTAG-3'). Genotyping of
otpam866 embryos was performed as described
(Ryu et al., 2007).
arnt2hi2639c embryos were genotyped using one primer pair
that crosses the viral insertion to detect the mutant allele (forward,
5'-ATACTGAGGGTGAACGCAGACG-3'; reverse,
5'TCGCTTCTCGCTTCTGTTCG-3'; A. Nasiadka, ZIRC, personal
communication) and a second primer pair flanking the viral insertion to detect
the wild-type allele (forward, 5'-GAACTGAGTTTGCGCGTTTGAGAC-3';
reverse, 5'-CGGAAATGTCGCTGTTGTTAGTTGTG-3')
(Golling et al., 2002).
Plasmids and probes
References for published probes used for expression analysis can be found
at
www.zfin.org.
For preparation of ddc (IRBOp991B1037D) and sim2
(IRBOp991C0138D) probes, cDNA clones from imaGenes/RZPD were used. For
generation of the crh, sim1a, trh probes, parts of the coding
sequences were PCR amplified and cloned (primer sequences are available on
request).
Full-length sim1a (ZFIN ID: ZDB-GENE-020829-1) and sim1b (si:ch211-152c2.1; ZFIN ID: ZDB-GENE-041210-5) were PCR amplified and cloned into pCRII using the following primers: sim1a forward, 5'-GCCGTAGAGCATGAAGGAGAAGT-3' and sim1a reverse, 5'-TCAGCTGCCATTGGTGATGA-3'; sim1b forward, 5'-GACGGGATTTTAACGCGATTAACG-3' and sim1b reverse, 5'-AAAGCGCGAGTGTTTCTCCGTCAG-3'.
sim1a was subcloned using the Gateway system
(Kwan et al., 2007) to yield
pDestTol2pA2;CMV/Sp6:sim1a-pA. sim1b was subcloned into
pCS2+ vector.
Whole-mount in situ hybridization, and fluorescent immunohistochemistry
were performed as described (Filippi et
al., 2007). TUNEL staining was carried out on whole mounts as for
whole-mount in situ hybridization. The TdT reaction (ApopTag Kit, Chemicon)
and detection with Anti-Dig-AP antibody (Roche) were carried out according to
the manufacturer's instructions.
Morpholino and mRNA injections
The following morpholinos were used (Gene Tools LLC): arnt2 (ATG),
5'-GGTTTACAGCGGCTGGTGTTGCCAT-3'; arnt2 (e2i2),
5'-TTCTCAGAAGAATTGCTCACCCCGC-3'; pou47 (ATG),
5'-ATGATTGGATGCTGTAGTCGCCATG-3': sim1a (e2i2),
5'-TGTGATTGTGTACCTGAAGCAGATG-3'; sim1b (ATG),
5'-CTCCTTCATCGTGCCGTTAATCGCG-3'; sim1b (e1i1),
5'-TATATATCTGACCTTGCGGGAACAC-3'; sim1b (e2i2),
5'-GCATCTTGCTGATTATTACCTGGAG-3'; sim1b (e4i4),
5'-TTTTCTATAAAGTGACTCACCCTGG-3'. The standard control,
p53 ATG, otpb splice and sim1a ATG morpholinos have
been described previously (Langheinrich et
al., 2002; Eaton and Glasgow,
2006; Ryu et al.,
2007). The published sim1a ATG morpholino did not work in
our hands and was substituted by a newly designed sim1a splice
morpholino. Efficiencies and function of unpublished splice morpholinos were
confirmed by PCR on cDNA derived from morpholino-injected embryos using the
following primers: arnt2 splice forward,
5'-ACAGAAACCGAGCAAAATCGCATC-3'; arnt2 splice reverse,
5'-GAAAATTTGCTGGGTCCCTCGC-3'; sim1a splice forward,
5'-CGGCGGGAGAAGGAAAACAG-3'; sim1a splice reverse,
5'-ACCACCGCACGTCAATCCTG-3'; forward,
5'-GAAGGAGAAATCAAAGACTGCGGC-3'; sim1b splice 1-3 reverse,
5'-TCAGTCCGGCGTTTCGCTTC-3'. Full-length capped mRNA was
synthesized from pDestTol2pA2;CMV/Sp6:sim1a-pA (Sp6 mMessage mMachine
Kit Ambion). sim1a mRNA (100-200 pg) was injected per embryo. Both
morpholinos and mRNAs were diluted in H2O containing 0.05% Phenol
Red and 0.05% rhodamine dextran and injected at the one-cell stage.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
However, we consider group 1 to be prethalamic, groups 2, 4, 5 and 6 to be
otp-dependent posterior tubercular/hypothalamic
(Ryu et al., 2007), and groups
3, 7 and preoptic DA neurons to be hypothalamic, based on molecular marker
expression (A. Filippi and W.D., unpublished). To quantify reduction of DA
cells in the ventral diencephalons, we performed cell counts of distinct
groups in m1055 mutants and heterozygous siblings. As DA neuron
groups 4 to 6 are located in direct proximity to each other, we combined these
groups for analysis. In m1055 mutants, reduced DA cell numbers were
observed only for groups 2 (82.0% reduction) and 4-6 (81.3% reduction),
whereas groups 1 and 3 were not affected
(Fig. 1E-F; see Fig. S1E in the
supplementary material). For group 7 neurons, no obvious differences were
apparent between wild-type and mutant embryos, and cells were not counted, as
these neurons just form at 3 dpf. In m1055 mutants, the number of
both dopa decarboxylase and dopamine
transporter/slc6a3-expressing cells was selectively reduced in
those ventral diencephalic groups in which th expression was also
affected (see Fig. S2A-D in the supplementary material). Thus,
arnt2m1055 affects not only th expression, but
multiple aspects of DA neuron differentiation.
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) did not
result in complementation of the m1055 DA phenotype (data not shown),
suggesting that Arnt2 function is disrupted in m1055. We sequenced
the complete arnt2 ORF in m1055 homozygous mutants and
wild-type siblings, and detected a point mutation (T to A) at nucleotide
position 1869 of the arnt2 transcript isoform arnt2b ORF
(Tanguay et al., 2000), which
leads to a premature stop after 622 amino acids
(Fig. 1H,I). The truncated
Arnt2 protein in arnt2m1055 of both major arnt2
transcript isoforms (arnt2b and arnt2c) therefore still
contains the conserved bHLH domain, as well as the PAS domains, but lacks the
essential transactivation domain at the C terminus
(Jain et al., 1994).
arnt2hi2639c contains a retroviral insertion 26 bp
upstream of the arnt2 translation start site, which leads to a
complete absence of the arnt2 transcripts
(Golling et al., 2002) (data
not shown) and thus it can be considered as a null allele. DA cell counts at 3
dpf in both arnt2m1055 and
arnt2hi2639c alleles showed similar phenotypic strength,
suggesting that arnt2m1055 also behaves as a null allele
(see Fig. S1A-E in the supplementary material). At 24 hpf, arnt2
transcript can be detected in the entire brain
(Andreasen et al., 2002) with
an enhanced expression in parts of the telencephalon and ventral diencephalon
(Fig. 1K). In m1055
mutants, arnt2 is strongly downregulated, suggesting a positive
autoregulatory loop (Fig. 1L).
Co-expression analysis of arnt2 and TH clearly indicated that
arnt2 is expressed in all TH immunoreactive DA cells affected by the
arnt2m1055 mutation at 36 hpf
(Fig. 1M,N).
We next injected translation blocking (ATG MO) and splice blocking
antisense morpholinos (splice MO) targeting arnt2 (see Fig. S5A,C in
the supplementary material). Injection of both morpholinos reduced the number
of DA cells in the ventral diencephalon in a similar way to m1055
(Table 1;
Fig. 3B,C,F,G; and data not
shown). It is known that p53-dependent cell death is induced by some
morpholinos through off-target effects
(Robu et al., 2007).
Co-injection of p53 and arnt2 morpholinos did not attenuate
the reduction of DA cells in arnt2 ATG or in splice MO-injected
embryos, suggesting that the reduction of DA cells is due to a specific
knockdown of arnt2 (Table
1; and data not shown). We next investigated the neural phenotypes
of arnt2m1055 mutant embryos in more detail. As the
reduction of DA neurons in arnt2m1055 could be due to
elevated cell death, we investigated apoptosis in the mutants by TUNEL. We did
not observe increased apoptosis in arnt2m1055 embryos
between 24 and 72 hpf (see Fig. S2E-H in the supplementary material; data not
shown). We also detected no significant changes in cell proliferation by
analyzing pcna and mcm5 expression in mutant embryos at 36
and 60 hpf (data not shown). At 32 hpf, we further analyzed the expression of
the following genes important for early forebrain regionalization and
patterning: dlx2, dlx4, fezl, foxa2, lim1, nkx2.1a, nkx2.2a, nkx5.1,
pou50 and shh (see Table S1 and Fig. S2I-Q in the supplementary
material). As we did not detect any significant changes, we conclude that lack
of Arnt2 function does not affect establishment of early forebrain
patterning.
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), Arnt2
may also require Sim1 as dimerization partner during the development of DA
neurons in the diencephalon of zebrafish. Searching the zebrafish genome
(Ensembl version Zv6), we noticed that two sim1 paralogs exist. One
is located on chromosome 16 (termed sim1 in Ensembl) and a second one
is located on chromosome 4 (termed si:ch211-152c2.1 in Ensembl).
ClustalW alignment of Sim1 proteins from several species show that the
sequence of the zebrafish predicted Sim1 protein shares 73% amino acid
identity with human SIM1 (see Fig. S3 in the supplementary material). We will
further refer to this sim1 gene as sim1a. Sim1a shares a
total of 54% identical amino acids with the second zebrafish Sim1 predicted
from si:ch211-152c2.1. The ORF of si:ch211-152c2.1 has only
53% amino acid identity with human SIM1 protein, and will be termed sim1b.
sim1a and sim1b are nearly identically expressed in the
embryonic diencephalon (see Fig. S4A-H'' in the supplementary material).
As the sim1b probe produced a stronger signal in fluorescent in situ
hybridization than did the sim1a probe, we decided to use
sim1b for co-expression analyses. At 1 dpf, most of the TH
immunoreactive (THir) cells belong to DA neuron group 2, whereas DA neurons of
group 3 are located closer to the midline
(Fig. 2B, arrowheads). At 1
dpf, sim1b is expressed in two major domains in the diencephalon and
midbrain (Fig. 2A,B). All THir
cells of group 2 expressed sim1b, whereas the THir cells of group 3
were located outside the sim1b-expressing domain
(Fig. 2B,D-D''). In most
embryos, we detected sim1b-positive non-THir cells
(Fig. 2D-D'', arrowhead)
intermingled with sim1b-positive/THir cells (arrows). Those cells
might either represent DA neuron precursors that are not fully differentiated
and have not yet started to express TH, or cells of another neuronal identity.
At 3 dpf, the expression domain of sim1b in the diencephalon had
separated into a smaller ventral domain in the preoptic region and a larger
rostrocaudally elongated domain slightly dorsal to it
(Fig. 2C). At this time point,
colocalization of sim1b and TH was clearly detectable in DA neuron
groups 2, 4, 5 and 6, whereas no colocalization was apparent in DA neuron
groups 1 and 3 (Fig.
2E-G'', arrowheads in E'',G''). Thus, sim1
is expressed in the same DA neurons, which are reduced in
arnt2m1055 mutants.
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50% (data not shown). In summary, our results demonstrate that
sim1a is required for specification of sim1-expressing DA
neurons, whereas sim1 non-expressing DA neurons are not affected in
sim1a morphants. Furthermore, the DA neuron reduction in
sim1a morphants is very similar to arnt2m1055
mutants, suggesting that Arnt2/Sim1 acts as a heterodimer to specify DA
neurons in the diencephalon of zebrafish.
arnt2m1055 mutants and sim1a morphants reveal a common function in neurosecretory hypothalamus
To characterize the defects in differentiation of other neuronal subtypes
in the hypothalamus of embryos lacking Arnt2 or Sim1 function, we analyzed the
expression of neuroendocrine hormones at 76 hpf
(Fig. 4; see also Table S1 in
the supplementary material). crh is expressed in various parts of the
embryonic zebrafish brain, including telencephalon, hypothalamus, posterior
tuberculum, thalamus, retina and hindbrain
(Fig. 4A). crh neurons
in posterior tuberculum and hypothalamus are intermingled with DA neurons of
the ventral diencephalic groups (Fig.
4Q,R) (Chandrasekar et al.,
2007). crh expression in arnt2m1055
mutants and sim1a morphants was strongly reduced or completely absent
for a small group of crh cells in the preoptic region. CRH neurons
adjacent to DA neurons in the posterior tuberculum/hypothalamus are reduced in
number, although we never observed a complete loss of these cells
(Fig. 4A-C, arrowheads). The
expression of trh in zebrafish has only been analyzed in adult brain
(Diaz et al., 2002). In 76 hpf
forebrain, trh is expressed in the preoptic region in a domain close
to the anterior commissure. In arnt2m1055 mutants and
sim1a morphants, trh expression in the preoptic area was
lost, whereas a domain in the hindbrain was unchanged
(Fig. 4D-F). A requirement for
sim1 in specification of zebrafish itnp- and
vsnp-expressing neurons has already been suggested
(Eaton and Glasgow, 2006;
Eaton and Glasgow, 2007). In
arnt2m1055 mutants, as in sim1a morphants,
itnp and vsnp neurons in the preoptic region were strongly
reduced or absent (Fig. 4G-M).
vsnp expression in the ventral hypothalamus was not affected in the
arnt2 mutants or sim1a morphants
(Fig. 4K-M). We frequently
observed itnp-expressing cells at ectopic locations posterior to the
domain in the anterior hypothalamus in sim1a morphants
(Fig. 4I, arrow), but not in
arnt2m1055 mutants. sst1 transcript was
detectable in diencephalon, midbrain and hindbrain
(Devos et al., 2002). Within
the diencephalons, sst1 cells are located in four distinct groups:
two in the preoptic region, one in the hypothalamus close to the postoptic
commissure, and one in the dorsal diencephalon. In both
arnt2m1055 mutants and sim1a morphants, only the
more dorsally positioned cell group in the preoptic region was strongly
reduced, whereas all other sst1 groups formed normally
(Fig. 4N-P). We demonstrated
that the reduction of neurosecretory cell types in sim1a morphants is
not caused by non-specific morpholino induced cell death using co-injection of
p53 MO to suppress non-specific apoptosis (see Fig. S6 and Table S2
in the supplementary material).
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; Acampora et al.,
1999; Ryu et al.,
2007). Ngn1 is involved in neurogenesis of diencephalic
DA neurons (Jeong et al.,
2006) and elavl3 (huC) is a marker of
differentiated neurons (Kim et al.,
1997). Early-born sim1b/THir DA neurons were located
adjacent to a diencephalic ngn1 expression domain, suggesting that
ngn1 expression is already shut off in prospective DA neurons before
sim1a expression is turned on (see Fig. S7A-A''' in the
supplementary material, arrowheads). Furthermore, all
sim1b/TH-positive cells co-express elavl3, indicating that
Sim1 acts late during DA neuron differentiation only (see Fig.
S7B-B''' in the supplementary material). During specification of a
subset of neurosecretory cell types in the mammalian hypothalamus, parallel
Sim1 and Otp regulatory input converges at the level of the common downstream
target pou3f2/brn2 (Wang and
Lufkin, 2000). In zebrafish embryos lacking either Arnt2/Sim1
function or Otp function, or both, we detected significant reduction of
pou47 (zebrafish homolog of mammalian pou3f2) only in the
preoptic area, whereas expression in the posterior tuberculum/hypothalamus is
only slightly reduced, if at all (see Fig. S8 in the supplementary material).
We attempted to analyze the role of pou47 by morpholino knockdown;
however, knockdown of pou47 did lead to severe brain malformations,
preventing evaluation of pou47-specific defects (data not shown). In
summary, Arnt2 and Sim1 appear to act late in neuronal differentiation.
Combined overexpression of Sim1 and Otp induces differentiation of supernumerary ectopic DA and CRH cells in the posterior tuberculum/hypothalamus
We next analyzed whether Sim1a/Arnt2 and Otpa are sufficient to induce
ectopic DA or neurosecretory cell types. It has already been shown that
ectopic Otpa can induce some aspects of DA cell identity upon mosaic
expression by plasmid injection under the control of the CMV promoter
(Ryu et al., 2007). Global
overexpression of otpa mRNA instead leads to strong embryonic
malformations owing to disturbed gastrulation
(Ryu et al., 2007). For late
stage-specific overexpression, we created a transgenic line for heatshock
inducible Otpa. Activation of otpa expression in hs:otpa
fish directly after gastrulation resulted in morphologically intact embryos,
which expressed otpa ubiquitously (see Fig. S9 in the supplementary
material). In contrast to otpa, injection of sim1a mRNA did
not distort embryonic development and could therefore be used in
gain-of-function studies. We did not overexpress arnt2, as it appears
to be expressed globally during early brain development.
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However, when we assayed th expression at 20 and 25 hpf in Otp/Sim1OE embryos, we detected a threefold increase in cell number compared with control embryos (Fig. 6A,B; Fig. 7A). OtpOE and Sim1OE embryos also showed an elevated number of th neurons compared with controls, albeit to a much smaller extent. Consistent with the elevated number of th cells the number of dat/slc6a3-expressing cells was also increased at 25 hpf in Otp/Sim1OE embryos. Therefore, the supernumerary th cells appear to form mature DA neurons (Fig. 7C). In support of this, all supernumerary DA cells expressed both sim1b and otpb, even if these cells were located far away of the normal sim1/otp-positive domains (see Fig. S11 in the supplementary material). The rostrocaudal diencephalic domain in which the DA neurons were formed was elongated in Otp/Sim1OE embryos (see brackets in Fig. 6). We monitored the expression of dlx2, nk2.1, pitx3 and shh in all four experimental situations at 24 hpf. We did not detect a change in expression pattern of the analyzed patterning genes in OtpOE, Sim1OE or Otp/Sim1OE embryos, showing that early forebrain patterning was not affected (see Fig. S10 in the supplementary material).
At 32 and 48 hpf, additional DA neurons of groups 4-6 form posterior and
ventral to group 2. We performed separate cell counts of anterior and
posterior DA neuron populations at these stages. At 32 hpf and 48 hpf, the
cell number of DA cells of group 2 in Otp/Sim1OE, OtpOE, Sim1OE and control
embryos was similar to 25 hpf, arguing that new group 2 cells did not form
during this time interval. At 48 hpf, group 2 DA cells were not tightly
clustered as in control embryos, but instead were more dispersed and occupied
ectopic locations in the ventral diencephalon
(Fig. 6E-F). The number of DA
cells of groups 4-6 was not altered in Otp/Sim1OE, OtpOE or Sim1OE embryos
compared with controls (Fig.
7B). For an additional Arnt2/Sim1- and Otp-dependent neuronal cell
type, we analyzed the expression of crh at 32 hpf. The first CRH
neurons appear only around 25 hpf
(Chandrasekar et al., 2007).
The number of CRH neurons was more than threefold increased in Otp/Sim1OE
embryos compared with control embryos (Fig.
6G-H; Fig. 7D). For
CRH neurons, the cell numbers in OtpOE and Sim1OE embryos were also slightly
elevated compared with control embryos
(Fig. 7D). These observations
indicate that Otp and Sim1 overexpression lead to supernumerary ectopic DA and
CRH cells in the posterior tuberculum/hypothalamus only within defined
diencephalic competence fields. The supernumerary ectopic DA cells display
features of mature DA neurons, including the typical caudal axonal projections
(see Fig. S11E,E'', arrowheads in the supplementary material).
|
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), and
an insertional mutation reported (Golling
et al., 2002); however, functions during zebrafish development
have not been analyzed so far. Genetic knockouts of Arnt2 in mice are
perinatally lethal (Hosoya et al.,
2001; Keith et al.,
2001). Arnt2 functions both in developmental and physiological
processes in mammals (Gu et al.,
2000; Kewley,
2004). Despite the broad expression of arnt2, we did not
observe gross abnormal patterning or morphological malformations during
embryonic brain development of arnt2 mutants. Homozygous
arnt2 zebrafish are viable during embryonic and early larval stages,
but die eventually later than 5 dpf. The late lethality of arnt2
zebrafish mutants makes them a well-suited tool for studying Arnt2 function
during vertebrate development and physiology.
|
). Thus,
Arnt2, Sim1 and Otp are each required for specification of the same set of DA
subgroups in the zebrafish brain. Arnt2-, Sim1- and Otp-dependent neurons in
the anterior hypothalamus of mice do not differentiate terminally but instead
either die or change fate in the absence of any one of these transcription
factors. In arnt2 mutant the expression domains of otpa/b
and sim1a/b in the precursor territory are not altered at least until
3 dpf. Thus, DA progenitors expressing both Otp and Sim1 may continue to exist
until 3 dpf but fail to complete differentiation in the absence of Arnt2.
Whether these undifferentiated DA precursor cells subsequently die or adapt
other neuronal identities could not be determined.
Arnt2, Sim1 and Otp have been demonstrated to act in genetic pathways that
specify distinct neurosecretory cells in the murine hypothalamus
(Michaud et al., 1998;
Acampora et al., 1999;
Michaud et al., 2000;
Hosoya et al., 2001;
Keith et al., 2001).
Similarly, expression of five neurohormones is strongly reduced or absent in
the preoptic region (PO) of arnt2 mutants and sim1
morphants. Not all the neurons in the PO depend on Arnt2/Sim1 function, as
only one of two SST-expressing groups in the PO is reduced and DA neurons form
normally in the PO. Combined with a recent study, which shows the requirement
of Sim1 for ITNP- and VSNP-producing cells in the PO
(Eaton and Glasgow, 2006;
Eaton and Glasgow, 2007), our
data establish that the specification of specific neurosecretory cell types of
the hypothalamus is largely conserved in zebrafish. The mammalian Otp and
Arnt2/Sim1 pathways eventually merge at the level of Pou3f2/Brn2. For CRH
differentiation in mouse it is known that Pou3f2/Brn2 can directly bind and
activate the crh promoter (Nakai
et al., 1995). Similarly, in Arnt2/Sim1- and Otp-deficient
zebrafish, pou47 expression in the preoptic region is strongly
reduced. We also detected a slight reduction of pou47 in the
hypothalamus, but could not directly evaluate a role in DA differentiation
because of widespread defects in pou47 morphants.
We examined the epistatic relationship between arnt2, otpa/b and sim1a/b in zebrafish. arnt2, otpa/b and sim1a expression appear to be established independently, as expression domains appear unchanged if Arnt2/Sim1 or Otp activity is eliminated. However, the expression of the second sim1 paralog, sim1b, was abolished in arnt2 mutants. This could reflect subfunctionalization of the two paralogous sim1 genes and loss of the control mechanisms for initiation of expression in sim1b. Regardless of the mechanism of sim1b regulation, we were not able to assign a function to Sim1b protein using morpholino knockdown. Given that Sim1a knockdown phenocopies the arnt2 phenotype, Sim1b protein may have been rendered nonfunctional since the teleostean genome duplication.
We investigated whether Arnt2/Sim1 together with Otpa may be sufficient to
direct neuronal differentiation. Global overexpression of Sim1 by mRNA
injection together with Otp from a heat-shock inducible transgene induces
supernumerary DA and CRH cells in the ventral diencephalon. In case of DA
neurons, the field competent to respond to Sim1a/Otpa overexpression is
rostrocaudally elongated compared with wild type, whereas in case of CRH it is
enlarged both rostrocaudally and dorsoventrally. Thus, Sim1a/Otpa-induced
specification of DA and CRH neurons depends on a competence area for
differentiation (Fig. 8).
Notably, the enlarged area of DA/CRH differentiation is not caused by an
altered pre-pattern of the forebrain in Otp/Sim1OE embryos, as judged from
normal expression of dlx2, nkx2.1a, pitx3 and shh at 24 hpf.
Supernumerary DA cells in Otp/Sim1OE embryos may contribute only to the
anterior DA neuron cluster (group 2), and not to posterior DA neuron groups
(groups 4-6), which appear shortly after group 2. This could be caused by a
short time period of sufficient Otpa and Sim1a levels during which DA neurons
of groups 4-6 might not have reached the proper state to respond to these
factors. The supernumerary DA neurons in Otp/Sim1OE embryos express
otpb as well as sim1b. This suggests that the maintenance of
otpb and sim1b expression may be caused by an
auto-regulatory mechanism after initiation of otpa/b and
sim1a/b expression. The fact that overexpression of Otpa or Sim1a
alone increased the number of DA and CRH neurons in a much less pronounced way
implies that each factor has a limited potential to drive DA neuron
differentiation by itself. In a previous experiment
(Ryu et al., 2007), we used
microinjection of large amounts of an overexpression plasmid for mosaic high
level ectopic expression of Otpa and detected ectopic TH expression. By
contrast, in the experiments here, heat shock-induced Otpa expression from a
transgene probably produced only physiological Otpa levels. A further
difference is that plasmid-driven mosaic expression provides Otp from early
stage on throughout random single cell lineages, whereas heat shock-induced
expression generates sufficient amounts of Otpa only in a time window
following heat shock. Therefore, the different outcome of the experiments is
probably due to different Otpa levels, arguing that the potential of Otpa to
drive neuronal differentiation independently or in combination with Sim1a may
be concentration and time dependent.
Interestingly, two other bHLH-PAS transcription factors have been
previously linked to a DA neuronal phenotype. Two neuronal PAS domain protein
members (NPAS1, NPAS3) are linked to schizophrenia-like behavior
(Erbel-Sieler et al., 2004), a
disease connected to DA dysfunction (reviewed by
Sesack and Carr, 2002).
Furthermore, the expression of TH is negatively regulated by NPAS1 in a mouse
DA neuronal cell line and NPAS1 might repress TH expression by directly
binding to the TH promoter (Teh et al.,
2007). These data suggest a more modulatory activity of NPAS1 on
TH expression levels in existing DA neurons rather than a role during DA
specification. Expression of Arnt2 (and Sim1) is also maintained during
adulthood in the paraventricular nucleus of mammals, making it possible that
these factors also modulate gene expression levels in mature neurons
(Michaud, 2001).
The di-mesencephalic DA systems of the substantia nigra/ventral tegmental
area are the most prominent DA systems in mammals. However, mesencephalic DA
neurons do not exist in zebrafish. Genetic analysis revealed that the largest
complement of zebrafish ventral diencephalic DA neurons is specified by Otp
(Ryu et al., 2007), whereas,
in mouse, dorsal hypothalamic and anterior pretectal A11 group DA neurons
depend on Otp function (Ryu et al.,
2007). As Arnt2, Sim1 and Otp are essential for specification of
the same populations of DA cells in zebrafish, one might suggest that
Arnt2/Sim1 could also play a role in A11 specification. Analyses of
sim1 expression domains in murine embryos indicate that sim1
may also be expressed in the region of A11
(Ema et al., 1996;
Fan et al., 1996).
It is interesting to speculate that the A11-type DA system may have
co-evolved with neurosecretory hypothalamic systems. A11-type DA neurons in
fish and mouse establish the diencephalospinal DA system
(Bjorklund and Skagerberg,
1979), but A11 neurons also have ascending projections and local
hypothalamic connectivity (Fuxe et al.,
1985; Takada et al.,
1988). Mammalian A11 DA neurons have been implicated in restless
legs syndrome (for a review, see Clemens et
al., 2006), a motor disorder that is characterized by abnormal
limb sensations and involuntary movement. CRH, OT, VP cells of the PVN and SST
cells of the APV also display descending projections to the brain stem and
spinal cord (Krisch, 1981;
Sawchenko, 1987;
Hallbeck and Blomqvist, 1999).
Thus, most Otp-specified neurosecretory cell types display diencephalospinal
projections in addition to local connectivity. Interestingly, VP circuitry has
been implied with sensorimotor function
(Rose and Moore, 2002), and
VP/OT neural circuits have been postulated to simultaneously regulate
somatomotor and autonomic systems (Kerman,
2008). Therefore, we hypothesize that Otp-specified neurons may
share a common function in control of motor activity patterns, which may have
evolved from an ancient motor control system at the base of vertebrate
evolution.
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/6/1007/DC1
* Present address: Max Planck Institute for Medical Research, Jahnstrasse 29,
69120 Heidelberg, Germany 
|
REFERENCES |
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TOP
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RESULTS
DISCUSSION
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