Neurogenin1 is a determinant of zebrafish basal
forebrain dopaminergic neurons and is regulated
by the conserved zinc finger protein Tof兾Fezl
Jae-Yeon Jeong*, Zev Einhorn*†, Sara Mercurio†‡, Susie Lee*, Billy Lau*, Marina Mione‡, Stephen W. Wilson‡,
and Su Guo*§
*Department of Biopharmaceutical Sciences, Programs in Human Genetics and Biological Sciences, University of California, San Francisco, CA 94143;
and ‡Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom
The development of vertebrate basal forebrain dopaminergic (DA)
neurons requires the conserved zinc finger protein Too Few (Tof兾
Fezl) in zebrafish. However, how Tof兾Fezl regulates the commitment and differentiation of these DA neurons is not known.
Proneural genes encoding basic helix–loop– helix transcription factors regulate the development of multiple neuronal lineages, but
their involvement in vertebrate DA neuron determination is unclear. Here we show that neurogenin 1 (ngn1), a vertebrate
proneural gene related to the Drosophila atonal, is expressed in
and required for specification of DA progenitor cells, and when
overexpressed leads to supernumerary DA neurons in the forebrain of zebrafish. Overexpression of ngn1 is also sufficient to
induce tyrosine hydroxylase expression in addition to the panneuronal marker Hu in nonneural ectoderm. We further show that
Tof兾Fezl is required to establish basal forebrain ngn1-expressing
DA progenitor domains. These findings identify Ngn1 as a determinant of brain DA neurons and provide insights into how Tof兾Fezl
regulates the development of these clinically important neuronal
types.
neurogenin 1 兩 pluripotent neural stem cell 兩 neurotransmitter
phenotype 兩 commitment and differentiation
T
he determination of neurotransmitter phenotype is an important aspect of neuronal differentiation, and in this regard,
dopaminergic (DA) neurons have attracted considerable attention because of their functional and medical importance (1).
Degeneration of substantia nigra DA neurons in humans is a
hallmark of Parkinson’s disease, and the malfunction of DA
neurons in other brain regions is implicated in psychiatric
disorders and neuroendocrine dysregulation. Therefore, understanding the determination of DA phenotype and the specification of DA neuronal circuitry may provide mechanistic and
therapeutic insights into these disorders. To date, only limited
number of known or putative transcriptional regulators, including Pax6, Dlx, Nurr1, Lmx1a, Lmx1b, Msx1, Foggy, and Too Few
(Tof兾Fez1), have been implicated in the specification of DA
phenotype in vertebrates (2–7, 35). Despite this knowledge, the
mechanisms leading to the early commitment of pluripotent
neural stem cells to DA lineage remain elusive.
The earliest DA neurons in zebrafish are detected at ⬇24 h
postfertilization (hpf) in the basal forebrain (8). They express
tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and dopamine transporter (DAT), a protein
involved in dopamine reuptake (9). Later during development,
these DA neurons have both ascending and descending projections, and are believed to be homologous to mammalian DA
neurons of both the basal forebrain and midbrain (10, 11).
Through forward genetic analysis, an adult viable zebrafish
mutant named too few (tofm808) has been isolated that displays
selective deficits of basal forebrain DA as well as adjacent
serotonergic (5HT) neurons (8). Molecular characterizations
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0600337103
have revealed that the too few mutant carries a point mutation
that changes Cys-287 to Ser in the second of the six zinc finger
motifs of the conserved zinc finger protein Fezl (Tof兾Fezl) (5).
Whereas these studies establish an important role of Tof兾Fezl in
DA neuron development, the mechanism by which Tof兾Fezl acts
in DA neuron specification is not clear.
The role of basic helix–loop–helix (bHLH) proteins in neural
development has been initially discovered in Drosophila (12) and
later studied in multiple vertebrate neuronal lineages (13, 14).
Neurogenins are a family of bHLH proteins related to Drosophila
Atonal (15). Multiple members of the Neurogenin family have been
discovered in mice (16, 17), and they play partially overlapping roles
in regulating neuronal specification. For example, Neurogenin1 and
Neurogenin2 are involved in the development of dorsal root ganglia
(18, 19), and Neurogenin2 is a determination factor for placodal
sensory neuron development (20). In zebrafish, only one neurogenin-like gene, named neurogenin 1 (ngn1), has been identified so far
(21). Ngn1 is required for the development of spinal, cranial
sensory, and epiphysial neurons (22–24).
Here we report that early-born basal forebrain DA neurons are
derived from Ngn1-expressing progenitor cells. We demonstrate
that Ngn1 is necessary for the development of these DA neurons,
and overexpression of Ngn1 leads to supernumerary DA neurons
in the zebrafish forebrain, and induces TH⫹ cells with an apparent
neuronal morphology on the yolk surface ectoderm. Furthermore,
we show that Tof兾Fezl is expressed in an overlapping fashion with
ngn1, and is required to specify ngn1-expressing DA progenitor
domains in the basal forebrain. Together, our data identify early
regulatory steps that lead to the commitment of pluripotent neural
stem cells to dopaminergic phenotype.
Results
Ngn1 Is Expressed in Dopaminergic Progenitor Cells. While examin-
ing genes that are expressed in the vicinity of DA neurons in the
basal forebrain of zebrafish, we noted that ngn1 is expressed in
the basal forebrain as distinct clusters before DA neuron appearance (Fig. 1 A and B), and later in close proximity to the
appearing nascent TH⫹ DA neurons (Fig. 1C). At 28 hpf,
between approximately three and six DA neurons were detected
in the basal forebrain on each side of the midline, and they were
the earliest born DA neurons in the entire zebrafish embryo (Fig.
1E). To determine whether these early-born basal forebrain DA
neurons are derived from ngn1-expressing progenitor cells, a
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: DA, dopaminergic; TH, tyrosine hydroxylase; hpf, hours postfertilization;
DAT, dopamine transporter; 5HT, serotonin; MO, morpholino.
†Z.E.
§To
and S.M. contributed equally to this work.
whom correspondence should be addressed. E-mail: suguo@itsa.ucsf.edu.
© 2006 by The National Academy of Sciences of the USA
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NEUROSCIENCE
Edited by Yuh Nung Jan, University of California School of Medicine, San Francisco, CA, and approved February 9, 2006 (received for review
January 12, 2006)
Fig. 1. ngn1 is expressed in DA progenitors. All images are lateral views of
anterior brain regions. Anterior is to the left, and dorsal is up. (A and B) ngn1
expression at 14- and 20-somite stages, respectively (arrows point to several
clusters in the ventral forebrain). (C) A 28-hpf embryo showing ngn1 expression (purple) in close proximity to TH⫹ DA neurons (red). (D–F) Confocal
images of 28 hpf ngn1-GFP transgenic embryos immunostained with GFP
antibody (green, D), TH antibody (red, E), and the merged image (F), showing
that GFP is detected in TH⫹ DA neurons. (G–I) High-magnification views of D–F.
(J–L) Confocal images of 28-hpf ngn1-GFP transgenic embryos immunostained
with GFP antibody (green, J), 5HT antibody (red, K), and the merged image (L),
showing that GFP is not detected in 5HT neurons. (M–O) High-magnification
views of J–L. di, diencephalon, t, telencephalon. (Scale bars, 64 ␮m in A and B,
60 ␮m in C–F and J–L, and 3 ␮m in G–I and M–O.)
GFP transgene driven by the ngn1 promoter (25) was used to
follow the fates of ngn1⫹ cells after they stop expressing ngn1,
because GFP protein is more stable than ngn1 mRNA, and
usually lasts ⬎6 h after the cessation of gfp transcript synthesis
(26). Because the first TH-immunoreactive (ir) DA neurons are
detected ⬇24 hpf (8), we would be able to observe that DA
neurons were GFP⫹ at 28 hpf, should they be derived from
ngn1-expressing progenitor cells. Therefore, double immunostaining with GFP and TH antibodies were carried out in 28 hpf
embryos, and the results revealed that all TH⫹ DA neurons were
positive for GFP at 28 hpf (Fig. 1 D–I, and Fig. 7 A–F, which is
published as supporting information on the PNAS web site).
Among GFP⫹TH⫹ neurons, some displayed strong GFP signal
that might correspond to newly born cells, whereas faint GFP
signals were detected in all other TH⫹ DA neurons that were
likely born at earlier times (Fig. 1I).
A group of serotonin (5HT) neurons (usually about one to two
5144 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0600337103
Fig. 2. ngn1 is required for DA neuron development in the basal forebrain.
All images are lateral views of anterior brain regions except I–L. Anterior is to
the left, and dorsal is up. (A–L) Immunostaining with TH antibody (A, B, G, and
L), in situ with TH (E and F), or DAT antisense probe (C and D), or immunostaining with 5HT antibody (M and N), shows that DA neurons in the ventral
forebrain is completely absent at 32 hpf, largely absent at 48 hpf, and remain
significantly defective at 72 hpf in ngn1-morphants, whereas TH⫹ DA neurons
appear normal in the retina, TH⫹ gut sympathetic neurons are normal in the
trunk region of ngn1 morphants, and 5HT neurons are not affected. (Insets)
Magnified views of basal forebrain 5HT neurons. DA, dopaminergic neurons;
DAT, dopamine transporter; di, diencephalon; epi, epiphysis; hb, hindbrain;
LC, locus coeruleus; mhb, midhindbrain boundary; t, telencephalon. (Scale
bar, 32 ␮m.)
neurons at ⬇28 hpf) develop in close proximity to DA neurons
in the basal forebrain (4). To determine whether ngn1-GFP is
also detectable in these neighboring 5HT neurons, we carried out
double immunostaining with GFP and 5HT antibodies. These
results indicate that these neighboring serotonergic (5HT) neurons were always GFP negative (Figs. 1 J–O and 7 G–L). This
observation indicates that the early-born basal forebrain DA
neurons are derived from ngn1-expressing progenitor cells, but
5HT neurons are probably not.
Ngn1 Is Required for the Development of Dopaminergic Neurons in the
Basal Forebrain. To determine whether ngn1 is required for DA
neuron development, we injected a ngn1 morpholino (MO) that has
Jeong et al.
been shown to effectively and specifically knockdown Ngn1 protein
expression (22, 23). Abrogation of Ngn1 activity by MO injection
led to a severe loss of DA neurons in the ventral forebrain (95% of
embryos showing none or few TH or DAT-positive cells by 48 hpf,
n ⫽ 54) (Fig. 2 A–D). Some residual TH staining was detected in
48 hpf ngn1 MO embryos, but the intensity was much reduced as
compared to control embryos (Fig. 2 A and B). DAT expression
appeared to be completely abolished in these embryos (Fig. 2 C and
D). This phenotype was confirmed in ngn1 mutants (see Fig. 6 G
and H) isolated from mutagenesis screens (24, 27). Examination of
an earlier developmental stage revealed that, at 32 hpf, DA neurons
were completely absent in Ngn1-defective animals (Fig. 2 E and F).
Examination of a later postembryonic larval stage show that, at 72
hpf, DA neurons appeared to recover somewhat but remained
significantly defective in the basal forebrain (estimated to be
⬇80–90% deficiency compared to control) (Fig. 2 G and H). The
overall brain patterning of ngn1 morphants was normal, and TH⫹
DA neurons in the retina (Fig. 2 I and J), TH⫹ noradrenergic
neurons of locus coeruleus (Fig. 2 A and B) and the sympathetic
ganglia (Fig. 2 K and L) appeared not significantly affected in the
ngn1 morphants. Moreover, the neighboring basal forebrain 5HT
neurons were not obviously reduced (Fig. 2 M, N, and Insets). The
lack of 5HT defects in the absence of ngn1 activity is consistent with
the lineage study showing that these 5HT neurons were not derived
from ngn1-expressing progenitor cells (Fig. 1 J–O). These data
suggest that ngn1 is required for the development of basal forebrain
DA neurons, but not for the development of neighboring 5HT
neurons and several TH⫹ neuronal groups in other regions of the
nervous system.
To determine the state of DA progenitor domains in the absence
Jeong et al.
Fig. 4. Misexpression of ngn1 leads to supernumerary DA neurons in the
forebrain and induces TH⫹ cells with an apparent neuronal morphology on
the yolk surface ectoderm. (A–H) Immunostaining with TH antibody (A–F) and
in situ with DAT antisense probe (G and H) shows that DA neurons are
significantly increased in the ventral forebrain and ectopically induced in the
telencephalon (Di DA neurons are out of focal planes in E–H) in ngn1-mRNA
injected embryos. (I and J) Immunostaining with TH antibody showing an
ectopic TH⫹ cell on the yolk surface ectoderm of ngn1-injected embryo. (K and
L) A high magnification view of (I and J), and Inset showing an ectopic TH⫹ cell
with an apparent neuronal morphology on the yolk surface ectoderm. DA,
dopaminergic neurons; DAT, dopamine transporter; di, diencephalon; LC,
locus coeruleus; mhb, midhindbrain boundary; t, telencephalon. (Scale bar, 15
␮m in K and L, 32 ␮m in A–H, and 64 ␮m in I and J.)
of ngn1 activity, we analyzed ngn1 expression in the ngn1 morphants. We observed strongly enhanced ngn1 RNA labeling in the
morphant embryos, possibly due to the fact that the ngn1 MO
inhibits the translation of ngn1 transcript, and somehow leads to an
enhanced transcript stability, although a negative feedback regulation of ngn1 transcription by Ngn1 protein is also a possibility.
Nevertheless, these analyses revealed that the basal forebrain
ngn1-expressing progenitor domains were intact in the ngn1 morphants at multiple developmental stages examined (Fig. 3), suggesting that, in the absence of ngn1 activity, progenitor domains are
formed but fail to commit and differentiate into DA fate.
Overexpression of Ngn1 Leads to Supernumerary DA Neurons in the
Basal Forebrain and THⴙ Cells with an Apparent Neuronal Morphology
on the Yolk Surface Ectoderm. Given that ngn1 is essential for DA
neuron development, we next determined whether overexpressing ngn1 has an impact on DA phenotype by microinjecting ngn1
PNAS 兩 March 28, 2006 兩 vol. 103 兩 no. 13 兩 5145
NEUROSCIENCE
Fig. 3. ngn1 expression in ngn1 morphants. Anterior is to the left, and dorsal
is up. ngn1 expression is higher in the ngn1 morphants, and Ngn1-expressing
progenitor domains are intact in the basal forebrain of ngn1 morphants at
various developmental stages as indicated.
Fig. 5. tof兾fezl is expressed earlier than ngn1 and later in overlapping
domains with ngn1 in the basal forebrain All images are lateral views of
anterior brain regions. (A–D) In situ hybridization with tof兾fezl cRNA probe (A
and C) and ngn1 cRNA probe (B and D). (E–H) Double in situ hybridization of
tof兾fezl (red)⫹TH (purple) (E and G), tof兾fezl (red) ⫹ ngn1 (purple) (F), and TH
(red) ⫹ ngn1 (pruple) (H). DA, dopaminergic neurons; di, diencephalon; t,
telencephalon. (Scale bar, 32 ␮m in E–H and 64 ␮m in A and D.)
mRNA into one- to eight-cell-stage zebrafish embryos. A proportion of ngn1 mRNA injected embryos (usually ⬍15%) displayed morphological deformity including malformed eyes and
brain (data not shown), as has been reported (21). Therefore, in
all mRNA injection experiments carried out in our study, only
the injected embryos that display grossly normal brain morphology were used to determine the impact of ngn1 misexpression on
DA neuron development. We found that misexpression of ngn1
at 100 ng兾ml led to a significant increase of DA neurons in the
basal forebrain (77% embryos showing a significant increase of
DA neurons, n ⫽ 64) (Fig. 4 A–H) and in some instances a few
ectopic DA neurons were detected in the telencephalon (13%,
n ⫽ 220, Fig. 4 E–H). Using the pan-neuronal marker HuC, we
observed that, as previously reported (21), the expression of
pan-neuronal marker HuC in the CNS was not grossly increased
by Ngn1 overexpression (data not shown). These observations
suggest that Ngn1’s ability in increasing DA neuronal subtypes
is not a consequence of an overall increased neuronal production
in CNS. Interestingly, we found that misexpression of ngn1 also
induced TH⫹ cells on the yolk surface ectoderm, albeit at a low
number and a low frequency (⬇10% of injected embryos have
about one to three TH⫹ cells on the yolk surface ectoderm, n ⫽
50) (Fig. 4 J and L), despite this, such ectopic TH⫹ cells were
never observed in control embryos (0% of control embryos have
TH⫹ cells on the yolk surface ectoderm, n ⫽ 211) (Fig. 4 I and
K). DAT-positive cells appeared not induced on the yolk surface
ectoderm by ngn1 misexpression at the concentration that we
tested (data not shown). These TH⫹ cells appeared to have a
neuronal morphology (Fig. 4 L and Inset). This observation is
consistent with the fact that ngn1 is shown to be capable of
inducing the pan-neuronal marker Hu on the yolk surface
ectoderm (21). Taken together, we conclude that overexpression
of ngn1 is able to increase the production of DA neurons in the
forebrain and induce TH⫹ neurons in nonneural ectoderm. It is
5146 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0600337103
Fig. 6. The requirement of tof兾fezl in establishing ngn1-expressing DA
progenitor domains. (A–D) ngn1 expression in 20-somite wild-type sibling and
the tof mutant embryos shows that ngn1 expression in the basal forebrain
(arrows) is reduced at 20 somites (A and B) but appears normal at 28 hpf (C and
D). (E and F) ngn1 expression is largely absent in the basal forebrain of tof兾fezl
morphant. (G and H) TH and tof兾fezl in situ (both purple) shows that, although
TH⫹ DA neurons are largely absent, the fezl expression appears normal in the
ngn1 mutant. di, diencephalon; t, telencephalon. (Scale bars, 32 ␮m in G and
H and 64 ␮m in A–F.)
worth noting that increased production of DA neurons was
largely detected in their endogenous location of the basal
forebrain, suggesting that Ngn1 requires additional factors in this
region to induce DA neuron production.
Tof兾Fezl Expression Overlaps with ngn1-Expressing DA Progenitor
Domains in the Basal Forebrain. Our loss- and gain-of-function
analyses indicate that Ngn1 is a crucial factor for basal forebrain DA
neurons. To explore how Ngn1 might be regulated in the development of DA neurons, we investigated the relationship between
tof兾fezl and ngn1. Tof兾Fezl was previously identified through zebrafish forward genetics as an essential factor for the development
of basal forebrain DA neurons; however, its mechanism of action
is unknown. In 10-somite (⬇14 hpf) embryos, tof兾fezl expression
was detected broadly in the forebrain (Fig. 5A), preceding the
detection of ngn1 expression in this region (Fig. 5B). At ⬇18 hpf,
whereas tof兾fezl expression remained broad in the forebrain (Fig.
5C), ngn1 expression became detectable in distinct clusters of
progenitor cells in the forebrain as well as other brain regions (Fig.
5D). During the period when DA neurons are specified, tof兾Fezl
expression became less broadly expressed and was detected in
telencephalon, as well as, in diencephalon (the basal forebrain),
close to but not within DA neurons (Fig. 5E). Double labeling
experiments showed that tof兾fezl expression domains encompassed
the ngn1-expressing domains in the basal forebrain (Fig. 5F). Both
tof兾fezl and ngn1 expression in these domains were down-regulated
by 48 hpf (Fig. 5 G and H). These analyses suggest that ngn1 and
tof兾fezl are coexpressed in progenitor domains that are in close
proximity to basal forebrain DA neurons.
The Basal Forebrain Expression of Ngn1 Is Transiently Reduced in the
too few Mutant and Is Abrogated in the tof兾fezl Morphant. Because
tof兾fezl expression is detected earlier than ngn1 in the forebrain
Jeong et al.
Discussion
Compared to some other neuronal types, our knowledge on the
development of vertebrate DA neurons is rather limited. The
involvement of these neurons in multiple human neurological
disorders including Parkinson’s disease, addiction, and schizophrenia makes it a worthwhile endeavor to understand the mechanisms
underlying their development. Here we provide in vivo evidence in
zebrafish for an important role of Ngn1 in basal forebrain DA
neuron development: Ngn1 is required for the development of
DA neurons, and when overexpressed, can lead to supernumerary
DA neurons in the forebrain and TH⫹ neurons on the yolk surface
ectoderm. Furthermore, we show that Tof兾Fezl, an evolutionarily
conserved zinc finger protein, is required to establish basal forebrain ngn1-expressing progenitor domains. These analyses identify
crucial early regulatory steps in the commitment of multipotent
neural stem cells to a dopaminergic lineage.
The Requirement of Ngn1 in DA Neuron Development. Expression
analysis, lineage study, as well as loss-of-function genetic analysis
establishes that ngn1 is expressed in the basal forebrain DA
neuronal progenitor cells and is required for the specification of
DA neurons. Because ngn1-expressing progenitor domains appear intact in the absence of Ngn1 activity, the cellular role of
Ngn1 is likely required within the progenitor cells. Because the
other neuronal types that the basal forebrain ngn1-expressing
progenitor cells may give rise to (we showed that the adjacent
5HT neurons are unlikely to be derived from these ngn1expressing progenitor cells and are not obviously increased in
ngn1-defective embryos) are unlikely to be known, the fate of
these basal forebrain ngn1-expressing progenitors in the absence
of Ngn1 activity remains to be determined. In addition, although
basal forebrain DA neuron defects remain in the postembryonic
larval brain, they appear to be less severe than earlier stages,
suggesting that additional mechanisms likely exist to regulate the
generation of DA neurons at later stages.
Ngn1 may play one or multiple of the following roles in DA
progenitor cells: it may regulate their proliferation or cell cycle exit,
regulate their acquisition of general neuronal properties, and兾or
regulate their acquisition of subtype identity, as proposed based on
studies of proneural genes in multiple other neuronal lineages (14).
Understanding the precise role of Ngn1 in DA neuron development
requires the understanding of Ngn1’s function at the molecular
level, for instance, the identification of Ngn1 target genes in the DA
neuronal lineage. The well known direct target of Ngn1, NeuroD,
Jeong et al.
is surprisingly not detected in the basal forebrain region in zebrafish
(J.-Y.J. and S.G., unpublished data), although the possibility exists
that perhaps other yet unidentified NeuroD-like genes are expressed in this region in zebrafish. Nevertheless, the requirement
and sufficiency of Ngn1 for the appearance of TH indicates that
Ngn1 may directly regulate DA neuron subtype-related genes. At
present, few genes are known to be involved in DA neuron
development in zebrafish. Although the differentiation of basal
forebrain DA neurons in mice is shown to require Pax6 and Dlx (6,
7), and the differentiation of midbrain DA neurons in mice is shown
to require the nuclear receptor Nurr1 (2, 28) and the LIM homeodomain transcription factor Lmx1b (3), the involvement of these
factors in the development of basal forebrain DA neurons in
zebrafish remains to be determined. Taken together, it will be of
great interest to test whether Ngn1 regulate these factors and兾or
other unidentified pathways during DA neuron determination.
The Role of Ngn1 in Inducing DA Neurons. Our gain-of-function
analysis demonstrates that Ngn1 is capable of inducing supernumerary DA neurons and a DA subtype-related gene in
nonneural ectodermal cells. These data strengthen the idea that
Ngn1 may be involved not only in specifying general neuronal
property, but also in specifying subtype identity in the DA
neuronal lineage. However, a definitive role of Ngn1 in DA
neuron subtype specification awaits future analysis assessing
whether Ngn1 can directly regulate DA subtype-related genes.
Although a few ectopic DA neurons are detected in the telencephalon, the most striking increase of DA neurons is restricted
within its endogenous location of the basal forebrain. In our view,
this observation has two implications. First, because more Ngn1
leads to more DA neurons in the basal forebrain, it suggests that
Ngn1 is a limiting factor in the development of basal forebrain DA
neurons. The nature of this limiting effect remains to be understood: Ngn1 may be a limiting factor in determining how many
progenitors are initially specified toward DA lineage, or it may be
a limiting factor in determining how many rounds of proliferation
can occur before DA progenitors can exit cell cycle and embark on
a differentiation pathway. Second, because DA neuron induction by
Ngn1 is largely restricted to the basal forebrain, it suggests that
Ngn1 requires additional factors that are present in the basal
forebrain to determine DA neurons, or its pro-DA activity is
inhibited in other regions of the nervous system.
Regulation of Ngn1 by the Conserved Zinc Finger Protein Tof兾Fezl.
Previous molecular genetic analysis using the too few mutant
zebrafish has revealed the importance of Tof兾Fezl, a conserved
zinc finger protein, in DA neuron specification (5, 8), but its
mechanism of action is unknown. The analyses carried out here
reveal that Tof兾Fezl controls DA neuron development in part by
establishing ngn1-expressing DA progenitor domains in the basal
forebrain. How does Tof兾Fezl specify ngn1-expressing progenitor domains? The fact that Tof兾Fezl protein contains six C2H2
zinc fingers suggests that it is a putative transcription regulator
and may directly or indirectly regulate ngn1 expression. Therefore, the identification of downstream target genes of Tof兾Fezl
will provide crucial insights into understanding its role in
specifying ngn1-expressing progenitor domains. In addition to
the zinc finger domain, Tof兾Fezl possesses a Groucho-TLE-like
repressor domain, which is found in transcription factors such as
TCF that can serve as both transcriptional activators and repressors. Thus, Tof兾Fezl may repress a factor that normally
inhibits the formation of ngn1-expressing progenitor domains;
alternatively, Tof兾Fezl may be involved in promoting progenitor
domains by activating the expression of ngn1.
A Homologous Role of Neurogenins in the Development of Zebrafish
Basal Forebrain DA and Mouse Mesencephalic DA Neurons. While our
work was being prepared for publication, it was reported that loss
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region and later overlaps with basal forebrain ngn1-expressing
progenitor domains, we determined whether tof兾fezl acts upstream
of ngn1 in regulating DA neuron development. We first examined
ngn1 expression in the tofm808 mutant, which results from a single
amino acid change in the Tof兾Fezl protein (5). The results showed
that ngn1 expression was reduced in the basal forebrain at ⬇20somite stage (Fig. 6 A and B), but was later found comparable to
the wild-type siblings at 28 hpf (Fig. 6 C and D). Because it is
possible that tofm808 mutation might be a hypomorphic allele, we
sought to determine whether the basal forebrain ngn1 expression
domains would be more severely affected in a case of stronger loss
of Tof兾Fezl activity. Therefore, we designed two splicing morpholinos that target different exon兾intron boundaries to knockdown
Tof兾Fezl activity (Fig. 8, which is published as supporting information on the PNAS web site). Knockdown of Tof兾Fezl with either
morpholino led to a severe defect of ngn1-expressing domains in the
basal forebrain, whereas ngn1 expressing domains in other brain
regions were largely unperturbed (Fig. 6 E and F). These analyses
suggest that tof兾fezl is required to establish ngn1-expressing progenitor domains in the basal forebrain. tof兾fezl expression in the
ngn1 mutant was largely normal as compared to the wild-type
siblings (Fig. 6 G and H), suggesting that ngn1 is not required to
regulate tof兾fezl expression.
of neurogenin 2 (ngn2) function in mice impairs the development
of mesencephalic DA neurons, whereas non-DA neurons in the
midbrain are unaffected (29). The mouse study and our work
presented here have two important implications: first, they
indicate that the role of neurogenins in DA neuron development
is evolutionarily conserved. Second, they show that basal forebrain DA neurons in zebrafish share common developmental
mechanisms with the mammalian midbrain DA neurons. Together with the dye-tracing experiments (10) and a functional
study (30), our work suggests that some basal forebrain DA
neurons in zebrafish are developmentally and functionally homologous to mammalian midbrain DA neurons.
One difference between the mouse study and our work is that,
although ngn2 is not sufficient to induce DA neurons in mice (29),
we show that ngn1 is capable of inducing DA neurons in zebrafish.
Thus, whereas ngn2 appears to have a permissive role in mouse
midbrain DA neuron development, ngn1 may have an instructive
role in basal forebrain DA neuron development in zebrafish. These
differences may be gene- and兾or species-dependent.
In conclusion, our study demonstrates an important role of
ngn1 in basal forebrain DA neuron development in zebrafish,
and moreover, reveals that the establishment of ngn1-expressing
DA progenitor domains requires the conserved zinc finger
protein Tof兾Fezl. Future analyses of these transcription regulators promise to unravel further mechanisms governing the
commitment and differentiation of multipotent neural stem cells
to a dopaminergic fate.
Analysis of Neuronal Phenotypes in Transgenic ngn1:GFP Embryos.
Twenty-eight-hpf Tg(-8.4ngn1:GFP) embryos were fixed overnight in 4% PFA in 0.1 M phosphate buffer (pH 7.4) and stored
in 100% methanol at ⫺20°C. Whole mount immunohistochemistry was performed as described (33). The following primary
antibodies were used: anti-GFP (Chemicon, monoclonal,
1:1,000), anti-TH (Chemicon, rabbit polyclonal, 1:1,000) and
anti-5HT (DiaSorin, rabbit polyclonal, 1:4,000). The following
secondary antibodies were used: anti-rabbit Alexa 633 (Molecular Probes, 1:200) and anti-mouse Alexa 488 (Molecular
Probes, 1:200). Fluorescent labeling was analyzed by laser scanning confocal microscopy.
MO and mRNA Injections and Analysis. The ngn1 MO was synthesized and injected as described (22, 23). For misexpression
experiments, capped RNAs from pCS2-␤-gal and pCS2-ngn1
plasmids were synthesized and injected at 100–800 ng兾␮l with
2–3 nl into the yolk of one- to eight-cell-stage embryos as
described (34). For details, see Supporting Text, which is published as supporting information on the PNAS web site.
In Situ Hybridization and Immunohistochemistry. RNA In situ hy-
bridization and immunohistochemistry were performed as
described (8).
Fish Stocks and Maintenance. Fish breeding and maintenance were
performed as described (31). Embryos were raised at 28.5°C and
staged according to Kimmel et al. (32). Fish heterozygous for the
ngn1hi1059 and tofm808 mutations were bred to obtain homozygous
embryos for analysis: ngn1 homozygous embryos were identified
by applying the ratio of 25% to a population (⬎50) of stained
embryos; tof mutant embryos were identified by genotyping for
the missense mutation (5).
We thank Drs. Frances Brodsky, Yuh Nung Jan, Bingwei Lu, and
John Rubenstein for their helpful comments on the manuscript;
Drs. Adam Amsterdam and Nancy Hopkins (Massachusetts Institute of
Technology, Cambridge) for the ngn1 mutant; Uwe Strähle and Patrick
Blader (University of Heidelberg, Heidelberg) for ngn1:GFP transgenic
fish; and Drs. Say-Yeob Yeo (National Institutes of Health, Bethesda),
Ajay Chitnis (National Institutes of Health, Bethesda), and Masahiko
Hibi (RIKEN, Tokyo) for plasmids. This work was supported by Searle
Scholars Award, Burroughs Wellcome Fund, and National Institutes of
Health grants (to S.G.), and by grants from the Wellcome Trust,
Biotechnology and Biological Sciences Research Council and European
Community (to S.W.W.). The financial support of Telethon–Italy Fellowship GFP03011 (to S.M.) is gratefully acknowledged.
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Materials and Methods
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