1 Laboratory for Vertebrate Axis Formation, Center for Developmental Biology,
RIKEN, Kobe, Hyogo 650-0047, Japan
2 Department of Molecular Oncology, Graduate School of Medicine, Osaka
University, Suita, Osaka 565-0871, Japan
3 Department of Frontier Biosciences, Graduate School of Frontier Biosciences,
Osaka University, Suita, Osaka 565-0871, Japan
4 Department of Biology, Chungnam National University, Daejeon 305-764,
Korea
* Author for correspondence (e-mail: hibi{at}cdb.riken.go.jp)
Accepted 29 January 2003
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SUMMARY |
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Key words: Homeobox, Proneural gene, Neurogenic gene, Zebrafish
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INTRODUCTION |
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After neural induction and patterning, neurogenic regions, the domains in
which neurogenesis takes place, are established at the gastrula or neurula
stages in amphibia and teleosts. The neurogenic regions are prefigured by the
expression of proneural genes, which function to promote the formation of
neurons. Many of these genes are homologues of the Drosophila
achaete-scute and atonal genes and encode basic helix-loop-helix
(bHLH) proteins. In zebrafish as well as in Xenopus, the proneural
gene neurogenin1 (ngn1; X-ngnr-1 in
Xenopus) is expressed in three longitudinal stripes on each side, in
which primary motor-, inter- and Rohon Beard (sensory-) neurons arise, at the
late-gastrula to early-segmentation (neurula) stage
(Blader et al., 1997;
Kim et al., 1997
;
Ma et al., 1996
). Proneural
genes, such as neurogenin, further induce downstream bHLH genes, such
as neurod, to elicit the transition from proliferative neural
precursor cells to postmitotic neurons, which express neuron-specific markers,
N-tubulin, and elav-related genes (including elav13/HuC)
(Blader et al., 1997
;
Kim et al., 1997
;
Lee et al., 1995
;
Ma et al., 1996
). In addition
to neural specification and determination, proneural genes also trigger the
process of lateral inhibition. Cells that highly express proneural genes
become neurons and simultaneously express the Notch ligands Delta or Serrate,
which activate Notch signaling in neighboring cells
(Chitnis and Kintner, 1996
;
Ma et al., 1996
). As a
consequence of the Notch signal activation, these neighboring cells express
repressors of neuronal differentiation that belong to the enhancer of
split-hairy (Hes/Her) family transcription factors and cease differentiating
to neurons, as is also proposed for Drosophila neurogenesis
(Chitnis et al., 1995
;
Wettstein et al., 1997
). In
zebrafish, the delta genes (deltaA, deltaB and
deltaD) are expressed in neurogenic regions and later in primary
neurons from the late gastrula stage (Appel
and Eisen, 1998
; Haddon et
al., 1998b
). The Hes/Her family genes her4 and
her9 are expressed in the neural plate, and her4 has been
shown to be regulated by the Notch signal and to inhibit neurogenesis in
zebrafish (Leve et al., 2001
;
Takke et al., 1999
). Mutations
in components of the Notch pathway in zebrafish, including deltaA and
notch1 (notch1a Zebrafish Information Network)
(deadly seven), lead to an increase in the numbers of
ngn1-expressing neural cells and an expansion of the primary neurons
within the three stripes (Appel et al.,
2001
; Gray et al.,
2001
). Furthermore, primary neurons are produced in very excessive
numbers in the mutant embryos of mind bomb (mib; previously
known as white tail), as observed in embryos expressing
dominant-negative Delta and Su(H) protein, which also functions downstream of
Notch (Appel and Eisen, 1998
;
Haddon et al., 1998a
;
Jiang et al., 1996
;
Schier et al., 1996
). It has
recently been reported that mib encodes a RING ubiquitin ligase that
is required for efficient activation of the Delta-mediated Notch signaling
(Itoh et al., 2003
). These
reports show that proneural genes activate a lateral inhibition program in
which Notch signaling is involved, which restricts the numbers of neurons in
zebrafish.
There are several genes that are reported to function downstream of neural
inducers and upstream of proneural genes in Xenopus and zebrafish.
These include the Xenopus Sox-related genes SoxD and
Sox2 (Kishi et al.,
2000; Mizuseki et al.,
1998b
), Zic-related genes Zic-r1 and
Zic3 (Mizuseki et al.,
1998a
; Nakata et al.,
1997
), and Iroquois genes
(Bellefroid et al., 1998
;
Gomez-Skarmeta et al., 1998
),
all of which can be regulated by the organizer-derived BMP inhibitors.
However, these genes are expressed homogenously in subdivisions or the entire
region of neuroectoderm, but not in the `neurogenic regions'. None of them has
been shown to be involved in the formation of posterior neurons.
Using an expression cloning strategy, we isolated a zebrafish homeobox gene, pnx, which is expressed in posterior neurogenic regions earlier than neurogenin1 and delta genes. Expression of pnx is initially regulated by the neural inducers and a posteriorizing signal from the non-axial mesendoderm; latterly, pnx expression in primary neurons is regulated by the Notch signaling. Gain- and loss-of-function studies of Pnx indicated that it is a transcriptional repressor that functions upstream of proneural genes to define posterior neurogenic regions. We found that Pnx functions downstream of the Notch signal. These data indicate that pnx is a novel repressor-type homeobox gene that functions in neurogenesis.
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MATERIALS AND METHODS |
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cDNA library construction and screening
Procedures for the cDNA library construction and screening were previously
published (Yamanaka et al.,
1998). Briefly, a cDNA library was constructed from LiCl-treated
(dorsalized) early gastrula embryos and inserted into a modified version of
pCS2+ (Turner and Weintraub,
1994
) (pCS2+SfiI). The bacteria transformants containing 200-300
cDNA clones each were pooled and 5'-capped RNAs were synthesized in
vitro from each pool. One to two nanograms of 5'-capped RNA was injected
into one- or two-cell stage embryos and the effects of the RNA injection were
evaluated by morphological inspection and in situ hybridization of the early
segmentation-stage embryos with the markers, six3 (a marker for
forebrain), engrailed3 (for the mid-hindbrain boundary),
krox20 (for rhombomeres 3 and 5) and deltaB (for primary
neurons). Zebrafish sax1 cDNA was isolated from a zebrafish early
gastrula cDNA library by low stringency hybridization using the pnx
cDNA as a probe. The nucleotide sequences for pnx and sax1
were deposited in the GenBank database under Accession Numbers AB067731 and
AB067732, respectively.
Transplantation
Transplantation of the ventrolateral marginal tissues of the zebrafish
early gastrula was performed as described
(Koshida et al., 1998;
Woo and Fraser, 1997
). The
yolk of one- or two-cell stage embryos was injected with 0.5% lysine-fixable
tetramethylrhodamine-dextran (2,000 kDa, Molecular Probes). When the embryonic
shield became apparent (shield stage, 6.5 hpf), the ventrolateral marginal
blastomeres were excised with a tungsten needle and then transplanted through
a glass micropipette into the animal pole of a sibling shield-stage
embryo.
Plasmid construction and RNA and morpholino injections
To construct the expression vector for EnR-fusion proteins (pCS2+EnR), the
repressor domain of Drosophila Engrailed (amino acid residues 1-226)
was amplified from pTB-En (Fan and Sokol,
1997) by PCR and inserted into the BamHI and
EcoRI sites of pCS2+. To construct expression vectors for
Pnx-
N, VP16-Pnx and EnR-Pnx, the cDNA fragment containing the amino
acid residues 35-182 of Pnx was amplified from pCS2+SfiI-Pnx by PCR
and subcloned into pCS2+, pCS2+NLSVP16AD (containing amino acid residues
412-490 of Herpes simplex virus protein I VP16) and pCS2+EnR.
To construct a plasmid for N-Pnx, the cDNA fragment of amino acid residues
1-34 of Pnx was amplified by PCR. To construct the expression plasmids for
GAL4DB (the DNA-binding domain of the yeast transcription factor Gal4)-fusion
proteins, the cDNA fragment of Pnx, Pnx-N, N-Pnx, EnR or VP16 was
subcloned into pSG424 (Sadowski and
Ptashne, 1989
). The cDNA fragments of the GAL4DB-Pnx fusion
protein were amplified from the pSG424 plasmids by PCR and inserted into
pEGFP-C1 (Clonetech). pCS3+MT zGroucho2 was constructed by inserting zebrafish
groucho2 (Takke and
Campos-Ortega, 1999
) into pCS3+MT.
To construct a plasmid for Myc-tagged green fluorescent protein (MTGFP), the NcoI and EcoRI fragment of pEGFP-C1 (Clonetech) was inserted into the pCS2+MT plasmid. The expression vector for Pnx-GFP was constructed by inserting the PCR fragment containing the 5'UTR and the coding region of pnx into the ClaI and NcoI site of the MTGFP plasmid.
Synthetic capped RNAs for Pnx, EnRPnx, VP16Pnx, Squint
(Rebagliati et al., 1998),
Fgf8/Ace (Furthauer et al.,
1997
), Dkk1 (Hashimoto et al.,
2000
), Antivin (Thisse et al.,
2000
), a dominant negative form of Xenopus Delta-1
(XDlstu) (Chitnis et
al., 1995
) and zebrafish Notch5ICD
(Itoh et al., 2003
) were
transcribed in vitro using the linearized plasmid DNA as a template, then
dissolved in 0.2 M KCl with 0.2% Phenol Red as a tracking dye, and injected
into one-cell-stage embryos using a PV830 Pneumatic PicoPump (WPI).
The antisense morpholino oligonucleotides were generated by Gene Tools
(LLC, Corvallis, Oregon). pnx MO for 5'UTR,
5'-CCTGtCGGTcACTTCaGAGAcGAGT-3'; control MO for 5'UTR,
5'-GAtTTGgTCGTTTCTTCcTGCtTCC-3' (lower case letters indicate
mispaired bases); pnx MO for the translational initiation site (TIS),
5'-GAATTGCTCGTTTCTTCGTGCATCC-3'; control MO for TIS,
5'-GAtTTGgTCGTTTCTTCcTGCtTCC-3'. The MOs were dissolved in
1x Danieau's buffer (Nasevicius and
Ekker, 2000) for the stock (10 mg/ml). For injection, the MO was
diluted in 1x Danieau's buffer to 0.4-2 mg/ml.
Whole-mount in situ hybridization, ß-galactosidase detection and
immunohistochemistry
Whole-mount in situ hybridization was performed principally as described
previously (Jowett and Yan,
1996). BM purple AP substrate (Roche) and Fast Red tablets (Sigma)
were used as a substrate for alkaline phosphatase. To prepare an antisense
pnx riboprobe, the EcoRI-XbaI fragment of
pnx cDNA was subcloned into pBluescriptSK+ (Stratagene) (pBSK+zPnx).
pBSK+zPnx was digested by EcoRI and transcribed with T3 RNA
polymerase. Antisense riboprobes in this study were generated as described:
ngn1 (Kim et al.,
1997
), krox20 (Jowett
and Yan, 1996
), hoxb1b
(Alexandre et al., 1996
),
elavl3/HuC (Kim et al.,
1996
), olig2 (Park et
al., 2002
), islet1
(Inoue et al., 1994
) and
islet2 (Tokumoto et al.,
1995
). sox19 cDNA
(Vriz and Lovell-Badge, 1995
)
was amplified by PCR and used to generate a riboprobe. Immunohistochemistry
was performed as described previously
(Fujii et al., 2000
).
Monoclonal antibodies, znp1, zn5 and zn12
(Trevarrow et al., 1990
) were
used at the concentrations 1:500, 1:200, and 1:500, respectively. Immune
complex was detected using the Vectastain ABC kit (Vector Laboratories), and
visualized in 0.7 mg/ml diaminobenzidine (Sigma) and 0.003%
H2O2. For the detection of ß-galactosidase
activity, embryos were fixed in 2.5% paraformaldehyde in PBS overnight at
4°C, rinsed three times for 10 minutes in PBS with 0.02% NP-40, and one
for 10 minutes with X-gal staining buffer (100 mM phosphate buffer at pH 7.3,
2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide) at 37°C for 1 hour, and fixed
again in 4% paraformaldehyde in PBS overnight at 4°C before in situ
hybridization. Photographs were taken using an AxioPlan-2 microscope (Carl
Zeiss) and HC-2500 3CCD camera (Fuji Film). Figures were assembled using Adobe
Photoshop, V6.0.
Reporter assay and immunostaining
For the luciferase reporter assay, human embryonic kidney (HEK) 293T cells
were transfected with the expression vectors for the Gal4-Pnx fusion proteins
(0.2 µg), pFR-Luc plasmid (1 µg, 5x GAL4 Binding Element
Luciferase, Stratagene) and pCS2+nßgal (0.2 µg,)
(Turner and Weintraub, 1994)
in the presence or absence of pCS3+MT zGroucho2 (2 µg). After a 36 hour
incubation, the cells were harvested and the luciferase activities were
measured. The ß-galactosidase activities were used for normalization of
the transfection efficiency.
For the immunostaining of cells, COS7 cells were transfected with pCS2+MT-Pnx. After a 24 hour incubation, the cells were fixed in 4% paraformaldehyde for 15 minutes, labeled with anti-Myc-epitope monoclonal antibody (1:200 dilution, 9E10, Sigma) and visualized using an Alexa Fluor 568-conjugated goat anti-mouse IgG secondary antibody (1:500 dilution, Molecular Probes).
Co-immunoprecipitation and western blot analysis
The various Pnx mutant constructs and the expression vector for Myc-tagged
zebrafish Groucho2 were co-transfected into 293T cells. Thirty-six hours after
transfection, the cells were lysed on ice in lysis buffer (20 mM Tris-HCl at
pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 4 mM PMSF,
10 mg/ml aprotinin). The lysates were immunoprecipitated with anti-GFP rabbit
polyclonal antibodies (MBL), separated by electrophoresis on a 4-20%
polyacrylamide gel, and transferred to a PVDF membrane. The membrane was
immunoblotted with an anti-Myc-epitope monoclonal antibody (1:5000 dilution,
9E10, Sigma). The immune complexes were visualized by a chemiluminescence
system (Renaissance; Dupont NEN Products).
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RESULTS |
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The number of pnx-expressing cells was increased and almost
homogenous within the three stripes in mind bomb (mib)
mutant embryos, in which the Notch signaling is perturbed
(Haddon et al., 1998a;
Itoh et al., 2003
;
Riley et al., 1999
), at the
early segmentation (neurula) stage (Fig.
3N), although the homogenous expression in the posterior
neurogenic regions at the mid-gastrula stage was not affected in the
mib mutant embryos (data not shown). A similar expansion of
pnx-expressing cells was detected in embryos expressing a
dominant-negative (DN) form of Xenopus Delta1
(XDlstu, Fig.
3P). By contrast, the pnx-expressing cells were severely
reduced in number or absent in the embryos expressing an activated form
(cytoplasmic domain) of zebrafish Notch5
(Fig. 3O). Similarly,
misexpression of ngn1, which elicits an increased or ectopic
expression of deltaA, deltaD and her4
(Takke et al., 1999
) (data not
shown), attenuated the pnx-expressing cells
(Fig. 3Q). All of these data
indicate that the initial induction of pnx expression is not
regulated by the Notch signal, but the spotty expression within the neurogenic
regions at the segmentation (neurula) stages is controlled by the Notch
signal.
Pnx acts as a transcriptional repressor
Pnx contains an Eh1 repressor domain. To address whether Pnx functions as a
transcriptional repressor, we constructed a series of Pnx mutant proteins that
could be expressed as fusion proteins with GFP and the DNA-binding domain of
the yeast transcription factor Gal4 (Fig.
4A). In Pnx-N, the N-terminal region containing the Eh1
domain was deleted. N-Pnx contained only the N-terminal region. EnR and VP16
were constructed with the repressor domain of Drosophila Engrailed
and the transcriptional activation domain of Herpes Simplex Virus type I VP16
(Sadowski et al., 1988
) for
positive and negative controls, respectively. Human 293T cells were
transfected with expression vectors for the Gal4-Pnx fusion proteins and the
luciferase reporter gene containing 5x Gal4-binding sites, and the
levels of Gal4-dependent transactivation and repression were determined
(Fig. 4B). Expression of the
wild-type Pnx and the EnR proteins repressed the basal transcriptional
activities. The deletion of the Eh1 domain (Pnx-
N) suppressed the
repressor activity of Pnx, while the N-terminal domain alone (N-Pnx) was
sufficient for the repressor activity. By contrast, the expression of the VP16
activation domain activated transcription. These data suggest that Pnx acts as
a transcriptional repressor and that the Eh1 domain in the N-terminal region
is essential and sufficient for the repressor activity.
|
Pnx is involved in the formation of posterior neurons
Injection of pnx RNA into one-cell stage embryos elicited the
expansion of primary neurons (data not shown), as observed when the RNA was
injected into two- or four-cell stage embryos. In addition to the neurogenic
phenotype, the ubiquitous pnx expression posteriorized the
neuroectoderm (the expression of forebrain-specific genes was abrogated and
the anterior hindbrain region was shifted to the anterior side) and often
induced abnormalities in the formation of axial mesendoderm (data not shown).
However, the reverse phenotypes of these non-neurogenic phenotypes were not
observed in the embryos in which the Pnx function was perturbed (described
below). These phenotypes might be artifacts associated with the misexpression
in non-ectoderm (see Discussion). Therefore, to analyze the specific effects
of pnx expression in the ectoderm, we injected pnx RNA
together with ß-galactosidase RNA into one blastomere of two- or four-
cell stage embryos, and examined the effects of pnx overexpression by
in situ hybridization with various genetic markers, when the
ß-galactosidase RNA was expressed in the ectoderm.
The misexpression of pnx in the posterior ectoderm elicited an
expansion and/or ectopic expression of the proneural gene ngn1 and
postmitotic neuronal marker elavl3/HuC within the domain expressing
ß-galactosidase [84% (95/112) for ngn1, 72% (78/108) for
elavl3, Fig.
5C,E,J,K]. The increased ngn1-and
elavl3-expressing cells were scattered within the neural plate and
never exhibited a homogenous expression pattern. The expression of the
Engrailed repressor domain (EnR) fusion protein of Pnx (EnRPnx) exhibited
similar effects on ngn1 and elavl3 expression to those seen
with Pnx [88% (37/42) for ngn1, 77% (34/44) for elavl3;
Fig. 5F,G]. The misexpression
of pnx in anterior neural plate reduced the ngn1 expression
(Fig. 5B, arrowhead). The
expression of the VP16-Pnx fusion protein strongly inhibited the expression of
ngn1 and elavl3 [89% (113/127) for ngn1, 94%
(86/92) for elavl3; Fig.
5H,I,L,M], supporting the repressor function of Pnx in
neurogenesis. These data suggest that pnx or pnx-related
genes are required for promoting ngn1- and elavl3-expressing
cells in the posterior neural plate. The misexpression of Pnx induced a slight
expansion of neural plate, which is marked by sox19 expression
(Vriz et al., 1996), but the
VP16-Pnx expression did not significantly affect the formation of neural plate
(Fig. 5O), suggesting that Pnx
does not have a strong neuroectoderm-inducing activity. The effect of the
pnx misexpression on the neural plate
(Fig. 5N) may be a secondary
effect accompanied by the expansion of primary neurons.
|
|
We further performed an epistatic analysis of pnx and the Notch signal. We injected VP16Pnx or Pnx MO into mib embryos or DN-Delta-expressing embryos. Expression of both ngn1 and elavl3 was decreased in these embryos, as in the VP16Pnx-exprssing embryos and Pnx morphant embryos [Fig. 7C, 65% (17/26); D, 74% (19/25); F, 97% (35/36); M, 71% (29/41); and N, 69% (30/43)], indicating that pnx is epistatic to the Notch signal.
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DISCUSSION |
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Roles of Pnx in the formation of posterior neurons
Injection of pnx RNA into one-cell stage embryos led not only to
the expansion of ngn1- and elavl3-expressing cells but also
to posteriorization of the neuroectoderm. We first thought that Pnx also
functions in the regionalization of the neuroectoderm. However, a loss of the
Pnx function, either by MO injection or by expression of VP16Pnx, which is
likely to inhibit the function of Pnx-related protein, did not anteriorize the
neuroectoderm (data not shown). Therefore, Pnx functions specifically in
neurogenesis but not in neural patterning, although premature neural
determination by ubiquitous Pnx expression might affect neural patterning.
Misexpression of pnx led to an expansion of ngn1- and
elavl3-expressing cells (Fig.
5). Loss of the Pnx function either by VP16Pnx expression or the
Pnx MO led to a reduction in ngn1- and elavl3-expressing
cells (Figs 5,
6). Because ngn1
functions upstream of the formation of elavl3-expressing postmitotic
neurons (Kim et al., 1997),
Pnx is likely to control the formation of primary neurons by upregulating the
proneural genes, such as ngn1. However, it was recently reported that
the inhibition of ngn1 function by MO leads to a reduction in
Rohon-Beard neurons but not primary motoneurons in zebrafish
(Cornell and Eisen, 2002
).
Similarly, targeted disruption of the ngn1 gene in mice does not
significantly affect the formation of motoneurons in the spinal cord
(Ma et al., 1998
). This could
be explained by the redundant functioning of Neurogenin-family genes.
Disruption of both the ngn1 and ngn2 genes in mice leads to
defects in the formation of motoneurons
(Scardigli et al., 2001
).
Another bHLH gene, olig2, which is specifically expressed in the
ventral spinal cord and is required for the formation of motoneurons in mice
and zebrafish (Mizuguchi et al.,
2001
; Novitch et al.,
2001
; Park et al.,
2002
; Sun et al.,
2001
), is a possible candidate that functions downstream of
pnx. However, olig2 expression was not affected in the
pnx MO-injected embryos (Fig.
6), suggesting Olig2 may function in parallel to Pnx. In
Drosophila, the homeobox gene vnd (ventral nervous system
defective) is involved in the development of ventral neuroblasts in the
central nervous system. vnd controls expression of the AS-C proneural
genes (Skeath et al., 1994
),
and also provides positional information for ventral neuroblasts
(Chu et al., 1998
;
McDonald et al., 1998
),
Although Vnd is close to Nkx2 but not Nkx1, the regulation of proneural genes
by homeobox genes is conserved between invertebrates and vertebrates.
Although pnx is expressed in three neurogenic regions (stripes),
VP16-Pnx-expressing embryos and Pnx morphant embryos displayed only a mild
reduction in Rohon-Beard neurons at the later stages (24 hpf embryo), compared
with the strong reduction in primary motoneurons. Furthermore, the effects of
Pnx MOs on the ngn1 and elavl3 expression were milder that
those of the VP16Pnx expression. These results suggest the existence of
molecules that function redundantly with Pnx. Pnx is a member of the Nkx1 gene
family, and displays homology with the Nkx1-family proteins Sax1 and Sax2
within their homeodomains. sax1 is expressed initially in the
posterior neuroectoderm in chick and mouse
(Schubert et al., 1995;
Spann et al., 1994
), like
pnx in zebrafish. Sax1, Sax2 or other Nkx1-related transcription
factors may work cooperatively with Pnx in the formation of motoneurons. As
described above, Pnx may also cooperate with Olig2, which functions downstream
of Hedgehog signals (Park et al.,
2002
), to promote the development of primary motoneurons.
Intriguingly, Olig2 is also reported to function as a transcriptional
repressor (Novitch et al.,
2001
; Zhou et al.,
2001
). Combinational repressions by Pnx and Olig2 may be required
for the primary motoneuron development. It remains to be elucidated how Pnx
cooperates with other factors to elicit the formation of interneurons and
Rohon-Beard sensory neurons.
Pnx promotes primary neurogenesis through activation of proneural
genes
As proposed for neurogenesis in Drosophila, in Xenopus
and zebrafish, proneural genes, such as Neurogenin genes and XASH3,
promote the expression of Delta genes, which activate Notch signaling in
neighboring cells and repress neural differentiation at least partly through
Hes/Her-family transcriptional repressors
(Chitnis and Kintner, 1996;
Ma et al., 1996
;
Takke et al., 1999
). The Notch
signaling and Hes/Her protein(s) further repress the expression of
ngn1 and its downstream genes that are required for neuronal
differentiation (Takke et al.,
1999
). This lateral inhibitory mechanism is implicated in
generating a restricted number of neurons in the neurogenic region. In this
report, we demonstrated that pnx also functions in the lateral
inhibition mechanism. First, gain and loss of Pnx function showed that
pnx regulates the expression of the proneural gene ngn1
(Fig. 4). Second, misexpression
of ngn1, which elicits an increased or ectopic expression of
deltaA, deltaD and her4
(Takke et al., 1999
) (data not
shown), led to a reduction in pnx-expressing cells
(Fig. 3). Finally,
pnx-expressing cells increased in number within the neurogenic
regions in the mib mutant embryos and DN-Delta-expressing embryos at
the segmentation (neurula) stages (Fig.
3), indicating that pnx expression is negatively
regulated by the Notch signal. These data support the idea that Pnx activates
proneural gene (such as ngn1)-dependent lateral inhibition machinery
that suppresses the expression of pnx in non-neuronal cells and
restricts the numbers of neurons (Fig.
7O).
How does Pnx promote neurogenesis?
Pnx contains an Eh1 repressor domain and interacts with the transcriptional
co-repressor Groucho2, at least in 293T cells. Reporter analysis revealed that
Pnx acts as a transcriptional repressor and that the Eh1-mediated interaction
with Groucho(s) is involved in this repressor activity
(Fig. 4). Furthermore, VP16-Pnx
functions as an antimorphic molecule in the formation of primary neurons.
These data indicate that Pnx functions as a transcriptional repressor and
should repress genes that have the ability to repress the proneural genes.
Candidates that are repressed by Pnx could include downstream components of
the Notch signal, such as the hes/her-family genes. However, this is
not the case. Misexpression of Pnx still increased the ngn1- and
elavl3-expressing cells in embryos in which Notch signaling was
suppressed. Furthermore, Pnx did not inhibit the expression of either
her4 or her9, which are the only Hes/Her-family members
reported to be expressed in the neural plate (data not shown). Furthermore,
inhibition of the Notch signal leads to an increase in the density of neuronal
cells `within the neurogenic region', but does not lead to the expansion of
neurogenic regions (Appel et al.,
2001; Chitnis and Kintner,
1996
). By contrast, the misexpression of Pnx in either wild-type
or mib mutant embryos elicited an `expansion' of the
ngn1-expressing neurogenic regions (Figs
5,
7). Furthermore, pnx
is epistatic to the Notch signaling in the formation of primary neurons
(Fig. 7), providing genetic
evidence that the pnx-mediated neurogenesis does not require the
Notch signal. These data indicate that Pnx can promote neurogenesis not by
inhibiting the Notch signal (lateral inhibition mechanisms), but rather by
expanding neurogenic regions within the neuroectoderm. To promote
neurogenesis, Pnx represses the expression of certain transcriptional
repressor(s), other than those downstream of the Notch signal, which inhibit
the proneural gene expression and neurogenesis
(Fig. 7O). The identification
of targets for Pnx will shed light on the mechanisms by which the neurogenic
regions are established and proneural genes are regulated.
In this study, we have demonstrated that Pnx is a novel transcriptional repressor that links posteriorizing signals to the initiation of a program for the posterior neurogenesis. The requirement of the repressor activity of Pnx provides a novel mechanism for neurogenesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexandre, D., Clarke, J. D., Oxtoby, E., Yan, Y. L., Jowett, T.
and Holder, N. (1996). Ectopic expression of Hoxa-1 in the
zebrafish alters the fate of the mandibular arch neural crest and phenocopies
a retinoic acid-induced phenotype. Development
122,735
-746.
Appel, B. and Eisen, J. S. (1998). Regulation
of neuronal specification in the zebrafish spinal cord by Delta function.
Development 125,371
-380.
Appel, B., Givan, L. A. and Eisen, J. S. (2001). Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development. BMC Dev. Biol. 1, 13.[CrossRef][Medline]
Barth, K. A., Kishimoto, Y., Rohr, K. B., Seydler, C.,
Schulte-Merker, S. and Wilson, S. W. (1999). Bmp activity
establishes a gradient of positional information throughout the entire neural
plate. Development 126,4977
-4987.
Bellefroid, E. J., Kobbe, A., Gruss, P., Pieler, T., Gurdon, J.
B. and Papalopulu, N. (1998). Xiro3 encodes a
Xenopus homolog of the Drosophila Iroquois genes and functions in
neural specification. EMBO J.
17,191
-203.
Blader, P., Fischer, N., Gradwohl, G., Guillemont, F. and
Strahle, U. (1997). The activity of neurogenin1 is
controlled by local cues in the zebrafish embryo.
Development 124,4557
-4569.
Blumberg, B., Bolado, J., Jr, Moreno, T. A., Kintner, C., Evans,
R. M. and Papalopulu, N. (1997). An essential role for
retinoid signaling in anteroposterior neural patterning.
Development 124,373
-379.
Brewster, R., Lee, J. and Ruiz i Altaba, A. (1998). Gli/Zic factors pattern the neural plate by defining domains of cell differentiation. Nature 393,579 -583.[CrossRef][Medline]
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C. (1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375,761 -766.[CrossRef][Medline]
Chitnis, A. and Kintner, C. (1996). Sensitivity
of proneural genes to lateral inhibition affects the pattern of primary
neurons in Xenopus embryos. Development
122,2295
-2301.
Chu, H., Parras, C., White, K. and Jimenez, F.
(1998). Formation and specification of ventral neuroblasts is
controlled by vnd in Drosophila neurogenesis.
Genes Dev. 12,3613
-3624.
Cornell, R. A. and Eisen, J. S. (2002).
Delta/Notch signaling promotes formation of zebrafish neural crest by
repressing Neurogenin 1 function. Development
129,2639
-2648.
Cox, W. G. and Hemmati-Brivanlou, A. (1995).
Caudalization of neural fate by tissue recombination and bFGF.
Development 121,4349
-4358.
Doniach, T. (1995). Basic FGF as an inducer of anteroposterior neural pattern. Cell 83,1067 -1070.[Medline]
Durston, A. J., Timmermans, J. P., Hage, W. J., Hendriks, H. F., de Vries, N. J., Heideveld, M. and Nieuwkoop, P. D. (1989). Retinoic acid causes an anteroposterior transformation in the developing central nervous system. Nature 340,140 -144.[CrossRef][Medline]
Erter, C. E., Solnica-Krezel, L. and Wright, C. V. (1998). Zebrafish nodal-related 2 encodes an early mesendodermal inducer signaling from the extraembryonic yolk syncytial layer. Dev. Biol. 204,361 -372.[CrossRef][Medline]
Fan, M. J. and Sokol, S. Y. (1997). A role for
Siamois in Spemann organizer formation. Development
124,2581
-2589.
Fujii, R., Yamashita, S., Hibi, M. and Hirano, T.
(2000). Asymmetric p38 activation in zebrafish: its possible role
in symmetric and synchronous cleavage. J. Cell Biol.
150,1335
-1348.
Furthauer, M., Thisse, C. and Thisse, B.
(1997). A role for FGF-8 in the dorsoventral patterning of the
zebrafish gastrula. Development
124,4253
-4264.
Gamse, J. and Sive, H. (2000). Vertebrate anteroposterior patterning: the Xenopus neuroectoderm as a paradigm. BioEssays 22,976 -986.[CrossRef][Medline]
Gomez-Skarmeta, J. L., Glavic, A., de la Calle-Mustienes, E.,
Modolell, J. and Mayor, R. (1998). Xiro, a Xenopus
homolog of the Drosophila Iroquois complex genes, controls
development at the neural plate. EMBO J.
17,181
-190.
Gray, M., Moens, C. B., Amacher, S. L., Eisen, J. S. and Beattie, C. E. (2001). Zebrafish deadly seven functions in neurogenesis. Dev. Biol. 237,306 -323.[CrossRef][Medline]
Grinblat, Y. and Sive, H. (2001). zic Gene expression marks anteroposterior pattern in the presumptive neurectoderm of the zebrafish gastrula. Dev. Dyn. 222,688 -693.[CrossRef][Medline]
Haddon, C., Jiang, Y. J., Smithers, L. and Lewis, J.
(1998a). Delta-Notch signalling and the patterning of sensory
cell differentiation in the zebrafish ear: evidence from the mind
bomb mutant. Development
125,4637
-4644.
Haddon, C., Smithers, L., Schneider-Maunoury, S., Coche, T.,
Henrique, D. and Lewis, J. (1998b). Multiple delta
genes and lateral inhibition in zebrafish primary neurogenesis.
Development 125,359
-370.
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu, T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000). Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation. Dev. Biol. 217,138 -152.[CrossRef][Medline]
Inoue, A., Takahashi, M., Hatta, K., Hotta, Y. and Okamoto, H. (1994). Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199,1 -11.[Medline]
Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust, D., Yeo, S. Y., Lorick, K., Wright, G. J., Ariza-McNaughton, L. et al. (2003). Mind bomb is a ubiquitin ligase that is essential for efficient activation of notch signaling by delta. Dev. Cell 4,67 -82.[Medline]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Jiang, Y. J., Brand, M., Heisenberg, C. P., Beuchle, D.,
Furutani-Seiki, M., Kelsh, R. N., Warga, R. M., Granato, M., Haffter, P.,
Hammerschmidt, M. et al. (1996). Mutations affecting
neurogenesis and brain morphology in the zebrafish, Danio rerio.Development 123,205
-216.
Jowett, T. and Yan, Y. L. (1996). Double fluorescent in situ hybridization to zebrafish embryos. Trends Genet. 12,387 -389.[Medline]
Kengaku, M. and Okamoto, H. (1995). bFGF as a
possible morphogen for the anteroposterior axis of the central nervous system
in Xenopus. Development
121,3121
-3130.
Kim, C. H., Ueshima, E., Muraoka, O., Tanaka, H., Yeo, S. Y., Huh, T. L. and Miki, N. (1996). Zebrafish elav/HuC homologue as a very early neuronal marker. Neurosci. Lett. 216,109 -112.[CrossRef][Medline]
Kim, C. H., Bae, Y. K., Yamanaka, Y., Yamashita, S., Shimizu, T., Fujii, R., Park, H. C., Yeo, S. Y., Huh, T. L., Hibi, M. et al. (1997). Overexpression of neurogenin induces ectopic expression of HuC in zebrafish. Neurosci. Lett. 239,113 -116.[CrossRef][Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Kishi, M., Mizuseki, K., Sasai, N., Yamazaki, H., Shiota, K.,
Nakanishi, S. and Sasai, Y. (2000). Requirement of
Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm.
Development 127,791
-800.
Kobayashi, M., Nishikawa, K., Suzuki, T. and Yamamoto, M. (2001). The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation. Dev. Biol. 232,315 -326.[CrossRef][Medline]
Koshida, S., Shinya, M., Mizuno, T., Kuroiwa, A. and Takeda,
H. (1998). Initial anteroposterior pattern of the zebrafish
central nervous system is determined by differential competence of the
epiblast. Development
125,1957
-1966.
Kudoh, T., Wilson, S. W. and Dawid, I. B. (2002). Distinct roles for Fgf, Wnt and retinoic acid in posteriorizing the neural ectoderm. Development 129,4335 -4346.[Medline]
Lamb, T. M. and Harland, R. M. (1995).
Fibroblast growth factor is a direct neural inducer, which combined with
noggin generates anterior-posterior neural pattern.
Development 121,3627
-3636.
Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N. and Weintraub, H. (1995). Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268,836 -844.[Medline]
Leve, C., Gajewski, M., Rohr, K. B. and Tautz, D. (2001). Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm. Dev. Genes Evol. 211,493 -500.[CrossRef][Medline]
Ma, Q., Kintner, C. and Anderson, D. J. (1996). Identification of neurogenin, a vertebrate neuronal determination gene. Cell 87,43 -52.[Medline]
Ma, Q., Chen, Z., del Barco Barrantes, I., de la Pompa, J. L. and Anderson, D. J. (1998). neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20,469 -82.[Medline]
McDonald, J. A., Holbrook, S., Isshiki, T., Weiss, J., Doe, C.
Q. and Mellerick, D. M. (1998). Dorsoventral patterning in
the Drosophila central nervous system: the vnd homeobox gene specifies ventral
column identity. Genes Dev.
12,3603
-3612.
McGrew, L. L., Lai, C. J. and Moon, R. T. (1995). Specification of the anteroposterior neural axis through synergistic interaction of the Wnt signaling cascade with noggin and follistatin. Dev. Biol. 172,337 -342.[CrossRef][Medline]
Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako, H., Nagao, M., Yoshida, S., Nabeshima, Y., Shimamura, K. and Nakafuku, M. (2001). Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31,757 -771.[Medline]
Mizuseki, K., Kishi, M., Matsui, M., Nakanishi, S. and Sasai,
Y. (1998a). Xenopus Zic-related-1 and Sox-2, two factors
induced by chordin, have distinct activities in the initiation of neural
induction. Development
125,579
-587.
Mizuseki, K., Kishi, M., Shiota, K., Nakanishi, S. and Sasai, Y. (1998b). SoxD: an essential mediator of induction of anterior neural tissues in Xenopus embryos. Neuron 21, 77-85.[Medline]
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. and Ericson, J. (2001). Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104,861 -873.[Medline]
Nakata, K., Nagai, T., Aruga, J. and Mikoshiba, K.
(1997). Xenopus Zic3, a primary regulator both in neural and
neural crest development. Proc. Natl. Acad. Sci. USA
94,11980
-11985.
Nasevicius, A. and Ekker, S. C. (2000). Effective targeted gene `knockdown' in zebrafish. Nat. Genet. 26,216 -220.[CrossRef][Medline]
Nguyen, V. H., Trout, J., Connors, S. A., Andermann, P.,
Weinberg, E. and Mullins, M. C. (2000). Dorsal and
intermediate neuronal cell types of the spinal cord are established by a BMP
signaling pathway. Development
127,1209
-1220.
Novitch, B. G., Chen, A. I. and Jessell, T. M. (2001). Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron 31,773 -789.[Medline]
Papalopulu, N., Clarke, J. D., Bradley, L., Wilkinson, D., Krumlauf, R. and Holder, N. (1991). Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos. Development 113,1145 -1158.[Abstract]
Park, H. C., Mehta, A., Richardson, J. S. and Appel, B. (2002). olig2 is required for zebrafish primary motor neuron and oligodendrocyte development. Dev. Biol. 248,356 -368.[CrossRef][Medline]
Piccolo, S., Agius, E., Leyns, L., Bhattacharyya, S., Grunz, H., Bouwmeester, T. and de Robertis, E. M. (1999). The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals. Nature 397,707 -710.[CrossRef][Medline]
Rebagliati, M. R., Toyama, R., Fricke, C., Haffter, P. and Dawid, I. B. (1998). Zebrafish nodal-related genes are implicated in axial patterning and establishing left-right asymmetry. Dev. Biol. 199,261 -272.[CrossRef][Medline]
Riley, B. B., Chiang, M., Farmer, L. and Heck, R.
(1999). The deltaA gene of zebrafish mediates lateral
inhibition of hair cells in the inner ear and is regulated by pax2.1.Development 126,5669
-5678.
Sadowski, I. and Ptashne, M. (1989). A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 17,7539 .[Medline]
Sadowski, I., Ma, J., Triezenberg, S. and Ptashne, M. (1988). GAL4-VP16 is an unusually potent transcriptional activator. Nature 335,563 -564.[CrossRef][Medline]
Sasai, Y. and de Robertis, E. M. (1997). Ectodermal patterning in vertebrate embryos. Dev. Biol. 182,5 -20.[CrossRef][Medline]
Scardigli, R., Schuurmans, C., Gradwohl, G. and Guillemot, F. (2001). Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord. Neuron 31,203 -217.[Medline]
Schier, A. F., Neuhauss, S. C., Harvey, M., Malicki, J.,
Solnica-Krezel, L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Stemple,
D. L., Rangini, Z. et al. (1996). Mutations affecting the
development of the embryonic zebrafish brain.
Development 123,165
-178.
Schubert, F. R., Fainsod, A., Gruenbaum, Y. and Gruss, P. (1995). Expression of the novel murine homeobox gene Sax-1 in the developing nervous system. Mech. Dev. 51,99 -114.[CrossRef][Medline]
Segawa, H., Miyashita, T., Hirate, Y., Higashijima, S., Chino, N., Uyemura, K., Kikuchi, Y. and Okamoto, H. (2001). Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish. Neuron 30,423 -436.[CrossRef][Medline]
Sharpe, C. R. (1991). Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system. Neuron 7,239 -247.[Medline]
Skeath, J. B., Panganiban, G. F. and Carroll, S. B.
(1994). The ventral nervous system defective gene
controls proneural gene expression at two distinct steps during neuroblast
formation in Drosophila. Development
120,1517
-1524.
Smith, S. T. and Jaynes, J. B. (1996). A
conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and
msh-class homeoproteins, mediates active transcriptional repression in vivo.
Development 122,3141
-3150.
Spann, P., Ginsburg, M., Rangini, Z., Fainsod, A., Eyal-Giladi,
H. and Gruenbaum, Y. (1994). The spatial and temporal
dynamics of Sax1 (CHox3) homeobox gene expression in the chick's
spinal cord. Development
120,1817
-1828.
Sun, T., Echelard, Y., Lu, R., Yuk, D. I., Kaing, S., Stiles, C. D. and Rowitch, D. H. (2001). Olig bHLH proteins interact with homeodomain proteins to regulate cell fate acquisition in progenitors of the ventral neural tube. Curr. Biol. 11,1413 -1420.[CrossRef][Medline]
Takke, C. and Campos-Ortega, J. A. (1999).
her1, a zebrafish pair-rule like gene, acts downstream of notch
signalling to control somite development. Development
126,3005
-3014.
Takke, C., Dornseifer, P., van Weizsacker, E. and Campos-Ortega,
J. A. (1999). her4, a zebrafish homologue of the
Drosophila neurogenic gene E(spl), is a target of NOTCH
signalling. Development
126,1811
-1821.
Thisse, B., Wright, C. V. and Thisse, C. (2000). Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo. Nature 403,425 -428.[CrossRef][Medline]
Tokumoto, M., Gong, Z., Tsubokawa, T., Hew, C. L., Uyemura, K., Hotta, Y. and Okamoto, H. (1995). Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish. Dev. Biol. 171,578 -589.[CrossRef][Medline]
Trevarrow, B., Marks, D. L. and Kimmel, C. B. (1990). Organization of hindbrain segments in the zebrafish embryo. Neuron 4,669 -679.[Medline]
Turner, D. L. and Weintraub, H. (1994). Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8,1434 -1447.[Abstract]
Vriz, S. and Lovell-Badge, R. (1995). The zebrafish Zf-Sox 19 protein: a novel member of the Sox family which reveals highly conserved motifs outside of the DNA-binding domain. Gene 153,275 -276.[CrossRef][Medline]
Vriz, S., Joly, C., Boulekbache, H. and Condamine, H. (1996). Zygotic expression of the zebrafish Sox-19, an HMG box-containing gene, suggests an involvement in central nervous system development. Mol. Brain Res. 40,221 -228.[Medline]
Wettstein, D. A., Turner, D. L. and Kintner, C.
(1997). The Xenopus homolog of Drosophila Suppressor of
Hairless mediates Notch signaling during primary neurogenesis.
Development 124,693
-702.
Woo, K. and Fraser, S. E. (1995). Order and
coherence in the fate map of the zebrafish nervous system.
Development 121,2595
-2609.
Woo, K. and Fraser, S. E. (1997). Specification
of the zebrafish nervous system by nonaxial signals.
Science 277,254
-257.
Woo, K. and Fraser, S. E. (1998). Specification of the hindbrain fate in the zebrafish. Dev. Biol. 197,283 -296.[CrossRef][Medline]
Yamanaka, Y., Mizuno, T., Sasai, Y., Kishi, M., Takeda, H., Kim,
C. H., Hibi, M. and Hirano, T. (1998). A novel homeobox gene,
dharma, can induce the organizer in a non-cell-autonomous manner.
Genes Dev. 12,2345
-2353.
Zhou, Q., Choi, G. and Anderson, D. J. (2001). The bHLH transcription factor Olig2 promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31,791 -807.[Medline]