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First published online 14 November 2007
doi: 10.1242/dev.011262
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1 Department of Molecular Cell Biology, Weizmann Institute of Science, PO Box
26, Rehovot 76100, Israel.
2 Division of Psychiatry Research, University of Zurich, August Forel-Str. 1,
Zurich CH-8008, Switzerland.
* Author for correspondence (e-mail: gil.levkowitz{at}weizmann.ac.il)
Accepted 19 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural development, Dopamine, Oxytocin
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Accordingly, developmental impairments in
hypothalamic differentiation are associated with defects in energy balance,
and in neuroendocrine and psychiatric disorders
(Swaab, 2004). For example,
loss of hypocretin neurons leads to narcolepsy, and patients with Prader-Willi
syndrome have a deficit in oxytocinergic (OT) neurons that is accompanied by
hyperphagia and severe obesity (Peyron et
al., 2000; Swaab et al.,
1995). The medial area of the hypothalamus contains magnocellular
and parvocellular neurons that control pituitary activities. The magnocellular
neurons project to the posterior pituitary where they release oxytocin and
arginine-vasopressin directly into the general circulation
(Landgraf and Neumann, 2004).
The parvocellular neurons affect the anterior pituitary by releasing
hypophysiotropic hormones such as dopamine and somatostatin into the
hypophysial-portal-vascular system
(Markakis, 2002).
Hypothalamic development poses a challenging model for understanding neural
patterning and specification because the hypothalamus contains multiple
nuclei, each composed of several neuronal cell types that form connections
with many parts of the nervous system
(Markakis, 2002). Uncovering
critical molecules regulating neural diversification of the hypothalamus is
essential to understand how this elaborate brain region is formed. Insights
into the differentiation of certain hypothalamic neurons has been contributed
by targeted gene knockouts of the transcription regulators Sim1, Brn2,
Arnt2, Hmx2/3 and Otp
(Acampora et al., 1999;
Michaud et al., 2000;
Michaud et al., 1998;
Schonemann et al., 1995;
Wang et al., 2004;
Wang and Lufkin, 2000). The
homeodomain-containing protein Orthopedia (Otp) is a key determinant
controlling the specification of neuroendocrine hypothalamic neurons
(Acampora et al., 1999;
Wang and Lufkin, 2000).
However, the signaling pathway(s) that regulate Otp and eventually lead to
synchronized hypothalamic differentiation have not been elucidated.
Here, we focus on studying the development of dopaminergic (DA) and OT-like neurons [termed isotocinergic (IT) neurons] representing mammalian parvocellular and magnocellular cell types, respectively. We report the mode of regulation of zebrafish Otpb during the development of these two prominent neuronal clusters. We show that regulated expression of Otpb by two novel converging pathways coordinate the development of IT and DA neurons.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Experiments were performed in accordance with the Weizmann Institute IACUC
protocol number 770104-1.
Plasmids and probes
Full-length otpb (see ZFIN ID: ZDB-GENE-990708-7 for nomenclature
history), pac1, and pacap1b cDNAs were amplified by PCR from
RNA that was isolated from embryos at 48 hours post fertilization.
Identification of the pac1 translation start site is detailed (see
Fig. S8 in the supplementary material). Activated pac1*
(E239Q) was generated by PCR-based site-directed mutagenesis. To construct the
1-5-pacap1b mutant, site-directed mutagenesis was used to
delete the cDNA nucleotides encoding amino acids 121-125 of the PACAP1b
precursor protein. cDNA was subsequently subcloned into either the
pCS2+ plasmid or the heat-shock response element (HSE)-driven
expression vector, pSGH2 (Bajoghli et al.,
2004) and confirmed by nucleotide sequencing. Oligonucleotide
primers that were used to amplify DNA templates for all digoxigenin
(DIG)-labeled probe synthesis reactions are described in
Table 1.
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). Otp
antibody was raised against a C-terminal Otp peptide and purified by affinity
chromatography as described (Lin et al.,
1999). Following in situ hybridization, embryos were embedded into
1.5% agarose, dehydrated and embedded in paraffin. Paraffin blocks were
sectioned (6 µm) on a microtome, mounted on slides and subjected to
deparaffinization. Microwave-induced Otp antigen retrieval was performed in 10
mM citric acid (pH 6) for 10 minutes. Sections were blocked in PBS with 20%
goat serum and 0.5% Triton X-100, and then incubated overnight at room
temperature with affinity-purified rabbit Otp antisera (at 2 µg/ml).
Sections were then washed with PBS and bathed with a goat anti-rabbit
biotinylated antibody (at 1:200 dilution) for 1.5 hours at RT and visualized
using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA).
Transient transfection, immunoblot and quantitative real-time PCR analysis
HEK293 cells were grown in 12-well plates and transfected (at 60%
confluence) with a total amount of 1.0 µg/well of the indicated pCS2-based
expression vectors using a standard calcium phosphate method. In some
experiments, cells were incubated for the indicated time periods in the
presence of different concentrations of a synthetic PACAP38 peptide
(Sigma-Aldrich, Rehovot, Israel). Proteins were harvested 24 hours post
transfection in 150 µl of hot SDS sample buffer and 15 µl of the crude
protein extract was fractionated by 10% SDS-PAGE followed by immunoblotting
with an affinity-purified anti-Otp antibody. Thereafter, PVDF membranes were
acid stripped and reprobed with a monoclonal anti-β-actin antibody (clone
AC-74, Sigma-Aldrich, Rehovot, Israel).
PC12 cell line (clone number CRL-1721, 7-10 passage) was obtained from the
ATCC Bioresource Center and propagated according to ATCC instructions.
Nuclear-enriched protein extraction as well as total RNA preparation were
previously described (Schreiber et al.,
1989). For western blot analysis of Otp from either PC12 or
zebrafish proteins, 20 µg total protein from each treatment was
fractionated on 8.5% SDS-PAGE and immunoblotted with anti-Otp antibody as
described above. Goat antisera directed against the nuclear regulator of
chromosome condensation 1 (Rcc1; Santa-Cruz Biotechnology, Santa Cruz, CA) was
used as an internal reference. Protein bands were quantified using an imaging
densitometer and analyzed with Multi-Analyst Software (Bio-Rad Laboratories,
Rishon Le Zion, Israel). [35S]methionine incorporation and
pulse-chase labeling procedures followed by anti-Otp immunoprecipitation and
gel-autoradiography were performed according to published methods
(Sambrook and Russell,
2001).
The level of endogenous otp RNA in PACAP38-treated PC12 cells was determined by quantitative real-time PCR kit (DyNAmo HS SYBR Green qPCR Kit, Finnzymes, Finland) using 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA). Two micrograms of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA) in a 20 µl reaction volume. PCR was performed in a 20 µl total reaction volume according to the kit instructions using rat-specific primers (Table 1) for either otp or βactin. Serial dilutions of the standard templates were also used for parallel amplification. The threshold cycles (Ct) were calculated and standard curves were plotted with Ct versus log template quantities. The quantities of samples were determined from the standard curves. otp levels were then normalized to those of β-actin in each corresponding sample.
Microinjection of morpholinos and RNAs
Capped RNAs were synthesized with mMESSAGE mMACHINE kit (Ambion, Austin,
TX) from linearized pCS2+ plasmids. The sequences of antisense
morpholino oligonucleotides (Gene Tools, LLC, Corvallis, OR) targeted to
fezl, otpb and pac1 are listed in
Table 2. Splice-blocking
morpholinos to fezl (Exon2-Intron2), otpa (Exon2-Intron2)
and pacap1b translation start blocking morpholino were previously
described (Jeong et al., 2006;
Ryu et al., 2007;
Wu et al., 2006). fezl
otpb and pac1 RNAs were injected at the concentrations indicated
in Table 3 into embryos at the
two- to eight-cell stage. Injected embryos were allowed to develop at
28.5°C.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
wild-type (WT) embryos, IT neurons were readily detected in the neurosecretory
preoptic nucleus (NPO), which is analogous to the mammalian supraoptic nucleus
(SON) (Peter and Fryer, 1983),
whereas hypothalamic DA cells developed in several clusters located at the
basal plate posterior tuberculum (PT), adjacent to IT neurons
(Fig. 1A). These DA clusters
express the previously described (Puelles
and Rubenstein, 2003; Rohr et
al., 2001) hypothalamic markers nk2.1a/titf1a,
nk2.1b/titf1b and sim1 (N.B., H.N.-R. and G.L., unpublished
material).
We have previously characterized the zebrafish mutant, too few
(tof m808), in which the development of hypothalamic DA
neurons is significantly impaired because of a recessive mutation in the gene
encoding the Fezl zinc-finger-containing protein (fezl, also known as
fezf2 - ZFIN) (Levkowitz et al.,
2003). Simultaneous examination of IT and DA neurons revealed that
the development of IT cells was attenuated in tof m808
embryos, which also displayed a clear deficit in DA neurons
(Fig. 1B). However,
hypothalamic hypocretin- and somatostatin-secreting neurons differentiated
properly in the tof m808 mutant
(Fig. 1C-F). Thus, proper
development of both IT and DA neurons requires Fezl activity.
We sought to identify the molecular events underlying fezl/tof
m808 activity. As IT and DA are generated in the NPO and PT,
respectively, we performed an RNA in situ hybridization screen to identify
candidate hypothalamic molecules that are expressed in these two adjacent
hypothalamic nuclei (J.B. and G.L., unpublished material). We identified two
genes, the homeodomain-containing gene, otpb, and the
G-protein-coupled receptor, pac1 (also known as adcyap1r1 -
ZFIN), that displayed a discernible expression pattern in the developing
zebrafish hypothalamus (Figs 2,
3 and see Fig. S1 in the
supplementary material). The zebrafish otpb gene is one of the two
predicted orthologs of the mammalian Otp, and PAC1 is the predominant
high-affinity receptor for the pituitary adenylate cyclase activating
polypeptide (PACAP) neuropeptide (Vaudry
et al., 2000). We observed that both Otp and PAC1 are expressed in
the developing PT and NPO adjacent to or at the Fezl-expressing domains,
respectively, suggesting that these molecules may be involved in
differentiation of the two hypothalamic nuclei
(Fig. 2A-D and see Fig. S1 in
the supplementary material). Otp protein was detected later in terminally
differentiated DA and IT neurons (Fig.
2E,F). Although PAC1 was readily detected in TH+ DA
neurons at 24-36 hpf, its expression in the later appearing (44-48 hpf) IT
cells was nearly undetectable by the time these neurons underwent terminal
differentiation (see Fig. S2 in the supplementary material and data not
shown).
fezl regulates otpb, but not pac1 gene expression in the hypothalamus
As both Otp and Fezl are critical determinants of hypothalamic development
we examined whether Fezl might regulate otpb by comparing its
expression in WT embryos to tof m808 mutants and to
fezl knockdown embryos. tof m808 embryos
displayed a complete loss of otpb gene expression in the PT area, and
a slight reduction in otpb+ cells of the NPO
(Fig. 3B, n=70/70).
The expression of otpb in the ventral diencephalon and hindbrain was
unaltered in tof m808 mutants. Complete inactivation of
Fezl by injecting two independent splice-blocking morpholino oligonucleotides
into tof m808 (denoted tof
fezlMO) resulted in a stronger effect; markedly
diminished otpb expression was observed in the PT, NPO and ventral
diencephalon, but not in the hindbrain
(Fig. 3C, n=10/15).
The latter result suggests that the tof m808 allele of
fezl is a hypomorph that retains residual transcriptional activity.
In agreement with the stronger effect of fezl knockdown on
otpb expression, the tof m808 mutant allele
displayed a delay in IT development whereas tof
fezlMO embryos displayed a sustained loss of IT
neurons (Fig. 3G). Finally, the
expression of the G-protein-coupled receptor pac1 in the PT, NPO and
pituitary was not affected by gene perturbations of fezl/tof
(Fig. 3D-F, n=60).
Hence, the Fezl transcription factor specifically regulated the expression of
otpb at two distinct hypothalamic nuclei that produce IT and DA
neurons.
|
). Interestingly, otpbMO treatment had a stronger
effect on the posterior hypothalamic DA groups 3-6, whereas group 2 was less
affected (Fig. 4C and see Fig.
S3 in the supplementary material).
The G-protein-coupled receptor, pac1, was detected in the
zebrafish NPO and PT before and during the period of DA and IT differentiation
(Figs 2 and
3 and see Fig. S2 in the
supplementary material). This suggested that pac1 might be a good
candidate transducer of an extracellular cue, which may be involved in DA and
IT development. Indeed, knockdown of pac1 gene activity impaired the
development of both IT and DA neurons (Fig.
4F and see Fig. S3 in the supplementary material). As in the case
of otpbMO, inactivation of pac1 mainly affected hypothalamic
DA groups 3-6, implying a common genetic pathway shared by otp and
pac1 (Fig. 4B,C,F).
Similarly to the reported tof m808 phenotype
(Levkowitz et al., 2003;
Rink and Guo, 2004), DA cell
groups in the retina, telencephalon and pretectal diencephalon, as well as
TH+;Otp+ noradrenergic neurons of the locus coeruleus
(LC), were not affected by otpb or pac1 inactivation (data
not shown). Consistently with the pac1 knockdown phenotype, the
high-affinity ligand to PAC1, PACAP1b, was expressed in the NPO and PT, and
was found to be necessary for IT and DA development (see Figs S1,S3 in the
supplementary material). The pacap1b morphant, however, had a more
pleiotropic effect including diminution of mid-hindbrain boundary and
cerebellar structures, suggesting that PACAP1b may bind to additional
G-protein-coupled receptors other than PAC1
(Vaudry et al., 2000).
pac1, otp and fezl form a genetic network controlling hypothalamic differentiation
Our results thus far suggest that differentiation of IT neurons as well as
of discrete clusters of hypothalamic DA cells is regulated by the
transcription factors Fezl and Otp and by the G-protein-coupled receptor,
PAC1. We next performed a genetic epistasis analysis to reveal the
hierarchical interaction between fezl, otpb and pac1. For
gain of function of Otpb we first determined the dose of otpb RNA
(7-15pg) that could rescue the otpb morphant without affecting
patterning (Fig. 4D and data
not shown). In order to activate the PAC1-mediated signaling pathway, we
generated a constitutively active form of the PAC1 receptor
(Cao et al., 2000), denoted
PAC1*. This construct had no obvious effect on patterning even at
the highest dose used (Fig. 4H
and data not shown). Injection of pac1* mRNA rescued the
pac1 knockdown phenotype, but could not complement otpb
deficiencies, suggesting that Otp is acting downstream of PAC1
(Fig. 4E,H). Consistently, the
deficiencies in DA and IT neurons that were caused by injection of
pac1MO could be rescued by co-injection of mRNA encoding to the Otpb
protein (Fig. 4G). Rescue of
pac1 morphant by otpb mRNA was not due to upregulation of
endogenous PAC1 or its ligand PACAP1b (see Fig. S5 in the supplementary
material).
Although otpb expression was diminished in the DA-deficient
tof m808 mutant (Fig.
3), overexpression of otpb RNA could not rescue the
tof phenotype (data not shown). We hypothesized that this was due to
insufficient Otpb protein expression levels in hypothalamic precursors. We
thus attempted to rescue tof m808 by injection of
heat-shock-inducible expression vector, which drives either Otpb or
PAC1* together with a GFP tracer
(Bajoghli et al., 2004),
followed by selection of mosaic embryos expressing high GFP levels in the
hypothalamus (see Fig. 5F).
Using this system, we were able to obtain complete rescue of the tof
phenotype by gain of function of Otpb (Fig.
5C; n=9/11), but not of PAC1*
(Fig. 5D; n=0/12).
Taken together the above epistatic analyses show that otpb acts
downstream of both fezl and pac1 and that proper development
of DA and IT neurons requires the activation of two signaling pathways that
converge to coordinate otpb function at different hypothalamic
nuclei.
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twofold increase) when Otpb was induced at 7
and 10 but not at 14 hpf (Fig.
5E-G). Accordingly, Otpb could rescue the tof phenotype
when its expression was induced at 7 but not 14 hpf
(Fig. 5C and data not shown).
As the majority of DA precursors exit the cell cycle by 14-16 hpf (N.
Russek-Blum and G.L., unpublished results) we conclude that Otpb is required
in hypothalamic DA progenitors and not in post-mitotic neurons.
PAC1 and its ligand PACAP regulate the levels of Otp protein
Although we demonstrated that otpb is epistatic to pac1,
knockdowns of pac1 and of pacap1b had no effect on
otpb transcript levels (Fig.
6C and supplementary material Fig. S4). Therefore, unlike the
evident effect of fezl/tof on the levels of otpb RNA
(Fig. 3A-C), regulation of
otpb transcription could not account for the genetic interaction
between pac1 and otpb. We then monitored Otp protein in
whole embryos following knockdown of either pac1 or its ligand,
pacap1b (Fig. 6 and
see Fig. S4 in the supplementary material). The levels of Otp protein were
moderately reduced in the hypothalamus and hindbrain following injection of
pac1MO (Fig. 6D;
n=25/30). Western blot analysis of Otp, which allows more accurate
quantification of the effects of pac1MO, resulted in a 50% decrease
in total Otp protein levels (Fig.
6D). As the anti-Otp antibody we used was raised against a
C-terminal epitope of Otp (see Materials and methods) it recognizes both Otpb
and its paralog Otpa. These two genes have nearly complete overlapping
expression domains (Ryu et al.,
2007) (data not shown). To demonstrate the net effect of PAC1 on
Otpb protein we compared Otp immunoreactivity in otpa morphants
(Fig. 6F; n=22) versus
pac1+otpa double morphants
(Fig. 6H). Otp immunoreactivity
was markedly reduced in the double morphant with no significant change in the
levels and in expression pattern of otpb RNA, suggesting that PAC1
might modulate otpb post-transcriptionally
(Fig. 6G,H; n=29/30).
To further examine this possibility we expressed zebrafish PAC1, PACAP1b and
Otpb in a heterologous cell culture system. In agreement with Lin et al.
(Lin et al., 1999), transient
expression of zebrafish Otpb in the human HEK293 cell line followed by western
blot analysis detected an Otp-immunoreactive protein band with an apparent
molecular mass of 50 kDa (see Fig. S4 in the supplementary material). Otpb
protein levels were increased two- to threefold after coexpression of Otpb
with PAC1*, the constitutively activated form of the receptor
(Fig. 7A). Higher induction
(sixfold) of Otpb protein was detected following coexpression of Otpb and PAC1
together with the PACAP1b precursor protein, but not with an N-terminally
truncated form of PACAP1b, denoted 1-5-PACAP, which acts as a PAC1
antagonist (Robberecht et al.,
1992) (Fig. 7A).
Hence, the in vivo genetic interaction between zebrafish pac1 and
otpb could be reconstituted in a mammalian system in vitro.
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).
Treatment of PC12 cells for up to 120 minutes with different concentrations of
a synthetic PACAP38 neuropeptide led to a fivefold increase of
endogenous levels of Otp protein (Fig.
7C,E). In accordance with our in vivo results, quantitative
real-time PCR analysis indicated that the level of otp RNA in PC12
cells was unaffected by PACAP stimulation, suggesting that a biochemical
pathway triggered by PACAP and its receptor PAC1 regulates Otp at the
post-transcriptional level (Fig.
7E). To determine the nature of this post-transcriptional control
we examined the ability of PACAP to affect Otp protein stability and
synthesis. Otp stability was examined by treating PC12 cells with the
translation inhibitor cycloheximide (CHX) and thereafter monitoring the levels
of Otp in the absence and presence of PACAP. This analysis showed that Otp
protein was relatively stable throughout the time of the experiment and that
PACAP-induced accumulation of Otp was blocked by CHX
(Fig. 7D,E). This result was
corroborated by [35S]methionine pulse-chase kinetics analysis of
Otp protein (see Fig. S9 in the supplementary material). To examine Otp
synthesis, we measured [35S]methionine incorporation into Otp in
the presence or absence of PACAP. The rate of CHX-sensitive
[35S]methionine incorporation into Otp was significantly increased
upon application of PACAP to PC12 cells
(Fig. 7F). Taken together the
above results indicate that PACAP controls the levels of Otp by promoting Otp
synthesis without affecting the stability of both protein and mRNA. In conclusion, we identified a novel regulatory network of cell-intrinsic and cell-extrinsic cues that act together to maintain coordinated development of hypothalamic DA and OT-like neurons.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tight regulation of Otp during hypothalamic development
It has been suggested that the intersection between the secreted molecules
Shh, Fgf8 and Bmp7 creates an induction site for hypothalamic DA identity
(Ohyama et al., 2005;
Ye et al., 1998). In spite of
these reports, the regulation of cell-autonomous determinants, which
presumably convert patterning signals into precise control of hypothalamic
development, is poorly understood. Otp is a critical cell-intrinsic
determinant, which controls the fates, migration and terminal differentiation
of mammalian hypothalamic neuroendocrine cells
(Acampora et al., 1999;
Wang and Lufkin, 2000). We
show that regulation of otp levels is important for the spatial and
temporal development of IT and DA neurons. We suggest that the tight
regulation of Otp is achieved by two sequential manners: first transcription
of otp mRNA is induced by Fezl, then Otp protein levels are modulated
by PAC1 (Fig. 8). In support of
this model, we show that the transcript levels of otpb were markedly
affected in the absence of fezl/tof gene function and that the levels
of Otp protein were controlled by PAC1 and its ligand PACAP (Figs
3 and
6). Our in vitro analyses show
that PACAP affects the rate of Otp protein synthesis, providing a mechanism
for the post-transcriptional control of otp, which was observed in
vivo (Fig. 7). Interestingly,
PACAP exerts a persistent post-transcriptional effect on the steady-state
levels of tyrosine hydroxylase in PC12 cells
(Corbitt et al., 1998).
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). The
varied sensitivities of otp, IT and DA to Fezl activity correlate
with fezl and otp expression patterns: whereas fezl
and Otp colocalize in the NPO where IT cells develop, only otp is
found in DA neurons of the PT (Fig.
2). Lack of Fezl-Otp colocalization in the PT is consistent with
our previously published mosaic analysis showing that fezl/tof
regulates hypothalamic DA development in a non cell-autonomous manner
(Levkowitz et al., 2003).
Hence, coordination of IT and DA cells may be attained by different
sensitivities of the otpb promoter to Fezl-mediated signal at
different hypothalamic territories.
Control of neuronal specification processes by Fezl
Recent studies described the phenotype of Fezl-deficient mice. Similarly to
zebrafish, mammalian Fezl is expressed in the telencephalon and diencephalon
(Hirata et al., 2004;
Mutsuga et al., 2005). In the
mouse telencephalon, Fezl is required for fate specification and axonal
projections of cortico-spinal motor neurons, subplate cortical neurons and
deep-layer pyramidal neurons (Chen et al.,
2005a; Chen et al.,
2005b; Hirata et al.,
2004; Molyneaux et al.,
2005). A close homolog of Fezl, denoted Fez, is expressed in the
mouse hypothalamus in partially overlapping domains with Fezl and
fez-fezl double deficient mouse displays defects in
diencephalic subdivisions (Hirata et al.,
2006a; Hirata et al.,
2006b). A similar role for zebrafish Fezl in diencephalic
patterning was also reported (Jeong et
al., 2007).
The precise regulation of Otp, DA and IT by the tof
m808 hypomorph (Figs
1 and
3) allows us to separate the
role of Fezl in regional patterning from its more selective role in cell
specification. The fact that Otpb could rescue the fezl/tof phenotype
indicates that Otpb is a critical target of Fezl that underlies its effect on
hypothalamic differentiation. In this respect, the proneural gene
neurogenin1 (neurog1) is regulated by Fezl and is both
necessary and sufficient for zebrafish DA development
(Jeong et al., 2006).
Similarly, dlx2, which controls specification of ventral thalamic DA
progenitors in the mouse, is regulated by Fezl
(Andrews et al., 2003;
Yang et al., 2001). Neither
neurog1 nor dlx2 is affected in either otpb or
pac1 morphants (data not shown), suggesting that Fezl may control
both early regional diencephalic commitment, which is mediated by Neurog1 and
Dlx2, and later cell-type specification, which is mediated by Otp
(Fig. 8). Notably, there are
two zebrafish orthologs of the mammalian Otp gene (denoted
otpa and otpb). Although this study demonstrates the
function and mode of regulation of otpb, a similar deficit in
diencephalic DA neurons was recently found in a null mutant allele of
otpa, suggesting that the activity of both otpa and
otpb is required for hypothalamic development
(Ryu et al., 2007). We found
that the transcription of otpa was affected in the absence of the
fezl/tof gene, reinforcing the significance of Fezl in regulating
hypothalamic cell fate decisions (see Fig. S5 in the supplementary
material).
Although the development of diencephalic neurons was not analyzed in
detail, hypothalamic neurons appear to be present in Fezl-/- mice
(Chen et al., 2005a;
Hirata et al., 2004). However,
in fezl/tof m808 mutants, otpb morphants and in
otp-deficient mice, selective groups of hypothalamic DA neurons are
reduced or missing, whereas other DA groups develop normally
(Rink and Guo, 2004;
Ryu et al., 2007)
(Fig. 4). Further analysis is
necessary to clarify whether subsets of hypothalamic neurons are affected in
fez/fezl-deficient mice.
Finally, activation of the Otp regulatory network might be relevant to
adult physiological states as fezl, pac1 and oxytocin are upregulated
in the rat SON in response to hyper-osmotic conditions and fezl and
oxytocin are downregulated following sustained hypo-osmolality
(Gillard et al., 2006;
Mutsuga et al., 2005).
Moreover, PACAP is enriched in rat mesencephalic DA neurons and protects DA
neurons from neurotoxin-induced death
(Chung et al., 2005;
Grimm et al., 2004;
Reglodi et al., 2004).
In sum, our data reveal two novel genetic pathways, which control Otp activity during differentiation of hypothalamic DA and OT-like neurons and may be relevant to hypothalamic developmental defects that cause metabolic and psychiatric clinical disorders.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4417/DC1
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