1 CNRS UMR 8542, Département de Biologie, Ecole Normale
Supérieure, 46, rue d'Ulm, 75230 Paris Cedex 05, France
2 Laboratoire de Génétique et Physiologie du Développement,
IBDM, CNRS-INSERM-Université de la Méditerranée, Campus
de Luminy case 907, 13288 Marseille Cedex 9, France
* Author for correspondence (e-mail: goridis{at}biologie.ens.fr)
Accepted 20 August 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Phox2b, Neurogenesis, Neural tube, Neuronal specification, Chick
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generic neuronal differentiation in vertebrates, as in Drosophila,
is regulated by basic helix-loop-helix (bHLH) transcription factors, which
promote it, and the Notch signaling pathway, which inhibits it. Several
vertebrate genes encoding bHLH proteins (such as Ngn1, Ngn2 and
Mash1) are thought to be equivalent to Drosophila proneural
genes and to confer competence to become a neuron (for a review, see
Kageyama and Nakanishi, 1997;
Brunet and Ghysen, 1999
;
Bertrand et al., 2002
).
Pioneering studies in Xenopus and zebrafish have demonstrated that
many of the neurally expressed bHLH factors promote neuronal differentiation
during primary neurogenesis (Ma et al.,
1996
; Blader et al.,
1997
). Recent work in mouse and chick embryos shows that they
function similarly in the neural tube of higher vertebrates
(Mizuguchi et al., 2001
;
Novitch et al., 2001
;
Scardigli et al., 2001
). A key
property of the early expressed bHLH proteins is that they restrict their own
proneural action by activating the expression of genes that inhibit
neurogenesis. This `lateral inhibition' has been well documented during
primary neurogenesis in Xenopus and in the chick retina, where neural
bHLH proteins up-regulate the expression of Delta that activates the Notch
receptor on neighboring cells (Chitnis et
al., 1995
; Henrique et al.,
1997
; Koyano-Nakagawa et al.,
1999
). Activated Notch in turn inhibits expression and activity of
the neural bHLH genes via its effector genes, the bHLH transcriptional
repressors of the Hes family, which have been shown to inhibit neurogenesis in
a variety of settings (Ishibashi et al.,
1995
; Wettstein et al.,
1997
; Kageyama and Nakanishi,
1997
; Kageyama and Ohtsuka,
1999
; Ohtsuka et al.,
1999
; Castella et al.,
2000
; Nakamura et al.,
2000
; Kondo and Raff,
2000
; Cau et al.,
2000
; Kabos et al.,
2002
).
Another class of molecules that have been implicated in the control of
neurogenesis are the Id HLH factors. Id proteins block differentiation and
promote proliferation in diverse cell types, including neural cells, mainly by
acting as dominant-negative inhibitors of positive regulatory bHLH proteins
(for a review, see Norton,
2000). The four vertebrate Id family members are expressed in the
embryonic neural tube in partially overlapping patterns
(Jen et al., 1997
). In mice
double mutant for Id1 and Id3, the neural tube shows signs
of premature neuronal differentiation
(Lyden et al., 1999
).
Conversely, overexpression of Id2 leads to overgrowth of the neural
tube (Martinsen and Bronner-Fraser,
1998
), and forced expression of Id1 or Id2
blocks neuronal differentiation (Cai et
al., 2000
; Toma et al.,
2000
). However, how the expression of the Id family members is
regulated in the neural tube and their precise mode of action in neural cells
have not been elucidated.
In general, newly born CNS neurons acquire phenotypes that reflect their
site of origin in the VZ. How this comes about has been best studied in the
ventral spinal cord. In response to a gradient of sonic hedgehog secreted from
ventral axial structures, the VZ is partitioned along the dorsoventral axis
into discrete domains that express particular combinations of homeodomain (HD)
transcription factors. These HD proteins appear to specify the subtype
identity of their neuronal progeny through the action of a different set of HD
proteins, which are switched on around the time of the last mitosis (for a
review, see Jessell, 2000;
Briscoe and Ericson, 2001
;
Lee and Pfaff, 2001
). Most of
the early expressed HD proteins appear to function as transcriptional
repressors and are thought to specify neuronal identity by repressing
alternative fates (Muhr et al.,
2001
; Vallstedt et al.,
2001
).
How the different molecular machineries that direct generic and
type-specific aspects of neuronal differentiation are coordinated in any class
of neurons remains poorly understood. One way this seems to be achieved is by
the fate-specifying action of proneural genes themselves. Rather than merely
drive a `generic' pathway of neuronal differentiation, they also participate
in the specification of neuronal types
(Fode et al., 2000;
Gowan et al., 2001
;
Scardigli et al., 2001
;
Parras et al., 2002
). A
striking recent example of the neural-fate determining properties of a bHLH
factor is provided by Olig2 function in spinal motoneuron progenitors
(Mizuguchi et al., 2001
;
Novitch et al., 2001
;
Lu et al., 2002
).
We have previously documented such a dual action on pan-neuronal and
type-specific differentiation for the fate-determining HD protein Phox2b. In
the ventral hindbrain, Phox2b is expressed by the progenitors of the
two main classes of cranial motor neurons, the branchimotor (bm) and
visceromotor (vm) neurons (collectively termed bm/vm neurons), and by their
postmitotic descendants, but not in somatic motor (sm) neurons
(Pattyn et al., 1997). In the
progenitors, Phox2b is necessary for cell cycle exit in proper
numbers. In the postmitotic precursors, Phox2b function is required
for all aspects of type-specific and generic differentiation. Conversely,
forced expression of Phox2b in the spinal cord promotes pan-neuronal
differentiation and emigration from the VZ and imparts a phenotype, which
resembles that of bm/vm neurons (Pattyn et
al., 2000
; Dubreuil et al.,
2000
). In the Phox2b-expressing cells, the early
postmitotic markers Delta1 and Math3/NeuroM are induced
prematurely, and the neurons thus generated ectopically express
Phox2a, choline acetyltransferase (ChAT) and
Islet1, but not Islet2, as do bm and vm neurons. However,
the molecular interactions by which Phox2b accomplishes this have not
been elucidated.
We have begun to examine the genetic interactions by which Phox2b promotes both, generic and type-specific aspects of neurogenesis. Ectopic expression studies provide evidence that Phox2b drives pan-neuronal differentiation by upregulating Ngn2 in the absence and of Mash1 in the presence of Nkx2.2 and by repressing the negative regulators of neurogenesis chick Hes5b and Id2. Initiation of a bm/vm fate represents a third activity that implies downregulation of Pax6 and Olig2 and upregulation of Nkx6.1 and Nkx6.2. Our data reveal how an HD transcription factor, through interaction with other factors expressed in the progenitor domain, coordinately regulates pan-neuronal and type-specific differentiation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electroporation
Chick embryos 44- to 52-hour-old (HH 12-14) were electroporated in ovo
essentially as described (Dubreuil et al.,
2000). The expression vectors were used at 1 mg/ml except for
Nkx2.2 at low doses and the homeobox fusion constructs, which were used at 0.5
mg/ml, and pCAGGS-AFP (0.8 mg/ml). We always co-injected
pCAGGS-AFP to visualize the transfected area. Embryos were allowed to
develop at 38°C for different time periods. After harvesting, the embryos
were fixed in 4% paraformaldehyde, embedded in gelatin and analyzed on
transverse neural tube sections at the transfected level.
Histological methods
Antisense RNA probes for Cash1
(Jasoni et al., 1994),
Delta1 (Henrique et al.,
1997
), EGFP (Clontech), cHes5b, Id2
(Martinsen and Bronner-Fraser,
1998
), Islet2
(Tsuchida et al., 1994
),
NeuroM (Roztocil et al.,
1997
), Ngn1 and Ngn2
(Perez et al., 1999
),
Nkx2.2 (Briscoe et al.,
1999
), Nkx6.1 (Qiu et
al., 1998
), Nkx6.2
(Cai et al., 1999
),
Olig2 (Zhou et al.,
2001
), Pax6 (kindly provided by T. Ogura), and
Phox2b and choline acetyltransferase (ChAT) (kindly provided by T.
Jessell) were labeled using a DIG-RNA labeling kit (Roche). In situ
hybridization and combined in situ hybridization and immunohistochemistry on
cryosections were carried out as described
(Hirsch et al., 1998
;
Dubreuil et al., 2000
). For
immunohistochemistry, the following antibodies were used: monoclonal anti-BrdU
(Sigma), monoclonal anti-Islet1/2
(Tsuchida et al., 1994
) and
rabbit anti-mouse Phox2b (Pattyn et al.,
1997
). BrdU incorporation and detection in chick embryos were
carried out as described (Sechrist and
Marcelle, 1996
). Pictures were taken with Kappa DX30, Nikon DXM
1200 or Leica DC300F CCD cameras using Kappa, ACT-1 or Leica software and
assembled using Adobe Photoshop.
Quantitative analyses
In situ hybridization signals were quantified by measuring the signal
intensity captured with a CCD camera on transverse spinal cord sections. On
each section, the mean signal intensity was recorded for the optimally
transfected area, as determined by GFP expression on an adjacent section, and
in an equivalent area from the non-transfected side. The results were
expressed as the difference in mean signal intensities between the transfected
and the non-transfected sides and statistical significance determined by
two-tailed t-test.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phox2b upregulates expression of proneural genes
One way by which Phox2b could drive neuronal progenitors to become
postmitotic is by inducing or boosting the expression of bHLH transcription
factors with proneural activity, which should promote pan-neuronal
differentiation. Ngn2 overexpression in neuroepithelial progenitors,
for example, has recently been shown to promote their exit from the cell
cycle, migration to the ML and expression of pan-neuronal markers
(Mizuguchi et al., 2001;
Novitch et al., 2001
). We show
in addition that, as does misexpression of Phox2b
(Dubreuil et al., 2000
),
misexpression of Ngn2 also upregulates Delta1, the earliest
known marker of postmitotic cells in the VZ
(Myat et al., 1996
)
(Fig. 1A). When overexpressed
in the chick neural tube, Mash1 behaved basically like Ngn2.
It promoted cell cycle exit as assessed by BrdU incorporation, Delta1
expression and relocation to the ML (Fig.
1C,D, and not shown). After transfecting GFP alone, most
electroporated cells were still in the VZ (not shown)
(Dubreuil et al., 2000
). Most
cells transfected with Ngn2 were positioned laterally already at 24
hours after electroporation (h.a.e.) (Fig.
1A), while after transfection of Mash1 and
Phox2b, an equivalent effect was observed only at 48 h.a.e.
(Fig. 1C,D)
(Dubreuil et al., 2000
). This
suggests that at the doses used, Ngn2 is more potent than
Mash1 or Phox2b.
|
Two observations made Cash1, the chicken ortholog of Mash1, an obvious
candidate for mediating the effect of Phox2b on pan-neuronal
differentiation. First, Mash1 is co-expressed with Phox2b in
the cranial bm/vm progenitors (Pattyn et
al., 2000) and is in fact the only bHLH gene we found expressed in
this progenitor population (M. R. H., unpublished). Second, in the absence of
Phox2b function, Mash1 expression in the bm/vm progenitors
is downregulated (Pattyn et al.,
2000
). However, misexpression of mouse Phox2b
(mPhox2b) did not induce Cash1 in the presumptive spinal
cord either at 6 or at 24 h.a.e. (Fig.
1E,G).
We thus reasoned that another bHLH factor with proneural activity might
mediate the effect of Phox2b in spinal regions of the neural tube.
Among the three genes studied (Cash1, Ngn1 and Ngn2),
Ngn2 was prominently expressed in the HH18-20 spinal cord, in a broad
ventrally located area, where the sm neurons are being born at this stage, and
in some scattered cells further dorsally. Misexpression of Phox2b
resulted in a marked dorsal expansion of the Ngn2 expression domain.
The increase in Ngn2 expression was greatest at 6 h.a.e. and declined
thereafter (Fig.
1F,F',I,H,H',J). Double-labeling with a Ngn2
probe and anti-Phox2b antibodies showed that most cells expressing
Ngn2 ectopically also expressed Phox2b indicating that
Phox2b functions cell-autonomously
(Fig. 1I). Cells in the
dorsalmost region of the neural tube consistently failed to respond to
Phox2b transfection with Ngn2 induction, in line with our
observation that Phox2b was also unable to induce early postmitotic
markers at this location (Dubreuil et al.,
2000). Ngn1 expression was upregulated by Phox2b
at 12 h.a.e. but not at 6 h.a.e. (not shown).
The failure to induce Cash1 could be explained by the requirement
for a co-factor present in bm/vm progenitors, but absent in the dorsal spinal
cord. Reasoning that Nkx2.2, the expression of which in the hindbrain
bm/vm progenitors coincides precisely with that of Phox2b
(Pattyn et al., 2000), may be
the missing factor, we co-transfected Nkx2.2 together with
Phox2b. This resulted in a marked induction of Cash1
throughout the transfected area at 20 but not at 6 h.a.e.
(Fig. 2A,A',B,B').
Nkx2.2 expressed alone at the same dose did not induce Cash1
(Fig. 2C,C'). At higher
concentrations, Nkx2.2 transfected alone also promoted Cash1
expression, but much more weakly than in combination with Phox2b
(Fig. 2D,D'). Hence, at
physiological expression levels, the combinatorial action of Nkx2.2
and Phox2b may be required to induce or maintain expression of
Mash1. These results raised the possibility that co-expression of
Phox2b and Nkx2.2 may also promote Ngn2 expression.
However, Ngn2 expression was repressed rather than activated by
transfecting Phox2b together with Nkx2.2
(Fig. 2B''), which can be
attributed to the negative regulation of Ngn2 in the spinal cord
(Zhou et al., 2001
).
|
Previously, we have shown that Phox2b misexpression in spinal
regions of the neural tube, where it is normally never expressed, promotes the
generation of neurons that migrate to the ML and induces aspects of a bm/vm
phenotype (Dubreuil et al.,
2000). This holds true also at hindbrain levels of the neuraxis,
where Phox2b-expressing vm neurons are born at this stage. At 48
h.a.e in the caudal hindbrain, most ectopically Phox2b-expressing
cells had relocated to the ML, an effect not seen after transfecting
GFP alone (Fig.
3A-B'). Most of them expressed Islet1/2
(Fig. 3C-C'') and the
bm/vm marker Phox2a, as do bm/vm neurons, but neither Islet2
nor Hb9, which are specific for sm neurons (not shown). Forced
expression of Phox2b also very efficiently induced the endogenous
Phox2b gene (Fig.
3B'), as it does at spinal levels (see
Fig. 5B''). We then
examined whether ectopic Phox2b would also upregulated Ngn2
when expressed alone and Cash1 when expressed together with
Nkx2.2. Misexpression of Phox2b in the caudal hindbrain
resulted in dorsal expansion of Ngn2 expression at 16 h.a.e., while
Cash1 was not induced (Fig.
3D-E'). By contrast, Cash1 was induced by
cotransfecting Phox2b together with Nkx2.2
(Fig. 3F-F'), but not
after transfecting Nkx2.2 alone at the same concentration
(Fig. 3G-G'). Clearly,
then, the effects of Phox2b misexpression on pan-neuronal and
type-specific differentiation can also be observed at rostrocaudal levels of
the neural tube, where Phox2b is expressed and known to be required
for the specification of vm progenitors.
|
|
These results suggest that in its normal expression territory in the ventral hindbrain, Phox2b promotes neurogenesis by upregulating Mash1 in combination with Nkx2.2. When misexpressed in more dorsal regions of the neural tube, it appears to do so by upregulating Ngn2 in cooperation with as yet unknown factors.
Phox2b inhibits expression of negative regulators of
neurogenesis
We next examined if Phox2b affects the expression of genes known
to play a role as negative regulators of neuronal cell cycle exit and
differentiation. Among such negative regulators, we focused on the Hes and Id
genes, which function as effectors of Notch signaling and as natural
inhibitors of bHLH factor activity, respectively
(Kageyama and Nakanishi, 1997;
Kageyama and Ohtsuka, 1999
;
Norton, 2000
).
Two members of the Hes family have been reported to be expressed in the
chick neural tube: Hairy1 and Hairy2
(Jouve et al., 2000). In the
early chick spinal cord, Hairy1 is expressed in a narrow dorsal
stripe and the floor plate (Jouve et al.,
2000
) and thus unlikely to function as negative regulator of
neurogenesis in the lateral neural tube. Strong expression of Hairy2
is confined to cells adjacent to the floor plate. Weaker expression is found
throughout the alar plate (Jouve et al.,
2000
), which was not affected by misexpression of Phox2b
(not shown). More recently, additional chick homologs of Hes genes have been
identified, which are most similar by sequence to mouse Hes5 (D.
Henrique, personal communication). Among them, chick Hes5b is
expressed in a broad lateral region of the neural tube (C. J., O.
Pourquié and D. Henrique, unpublished; see
Fig. 4L',N'). Like
mouse Hes5 (Kageyama and
Nakanishi, 1997
), chick Hes5b responds to activated Notch
and is upregulated in the chicken neural tube by expression of a
constitutively active Notch construct (D. Henrique, personal
communication). Hes5 also behaved as a negative regulator of
neurogenesis in the chick neural tube. Overexpression of mouse Hes5,
which we used to distinguish enforced from endogenous expression,
downregulated expression of the early postmitotic markers Delta1 and
NeuroM (Fig.
4C-D'). We then investigated whether Phox2b affected its
expression. Six hours after misexpression of Phox2b, we already
detected a decrease in chick Hes5b expression in the transfected area
in a sizeable fraction of the embryos (Fig.
4K,K'). At 24 h.a.e., chick Hes5b expression was
virtually extinguished on the transfected side of all embryos
(Fig. 4L,L').
|
Among the Id family members, we focused on Id2, the
overexpression of which has been found to cause overgrowth of the chick neural
tube (Martinsen and Bronner-Fraser,
1998). We found Id2 expression to be very dynamic over
the time period studied. Strong expression at early stages and in caudal
regions, where development is less advanced, was confined to the dorsal-most
neural tube with weak expression more ventrally. Upon further development,
expression became strong in a broad lateral domain
(Fig.
4K'',L'',N''). Id2 overexpression had
little effect on expression of Delta1
(Fig. 4G,G'), but
inhibited neuronal differentiation as judged from downregulation of
NeuroM (Fig.
4H,H'). As Delta1 precedes NeuroM in
postmitotic cells, this result may be taken to mean that Id2 affects
neurogenesis mainly at a step downstream of Delta1 expression. As in
the case of chick Hes5b, a slight decrease in Id2 expression
at 6 h.a.e. of Phox2b foreshadowed a massive repression at 24 h.a.e.
(Fig.
4K,K'',L,L'').
Our previous results have shown that ectopic Phox2b expression
promotes cell cycle exit (Dubreuil et al.,
2000). It was thus possible that repression of chick
Hes5b and Id2, which are expressed mainly in VZ progenitors,
was a mere consequence of cell cycle exit. Two types of evidence argue against
this explanation: (1) at 6 h.a.e., when we detected the first signs of
repression, equivalent numbers of BrdU-incorporating cells were present on the
transfected and the non-transfected side (not shown); and (2) double-labeling
for BrdU incorporation and chick Hes5b or Id2 expression at
18 h.a.e. showed that either gene was downregulated both in cells that had
incorporated BrdU and were thus in the S-phase of the cell cycle and in
BrdU-negative cells, which are a mixture of postmitotic precursors and of
progenitors in other phases of the cycle
(Fig. 4M). Hence,
downregulation of chick Hes5b and Id2 appears to precede
withdrawal from the cell cycle.
To provide evidence that inhibition of Hes and Id genes plays a role in mediating the neurogenesis-promoting activity of Phox2b, we co-expressed Hes5 or Id2 together with Phox2b. Co-transfection of Hes5 antagonized the increase in the number of Deltal-expressing cells observed after misexpression of Phox2b (Fig. 4E,E'). Quantification of the results showed that in the presence of mouse Hes5, the increase in Delta1+ cells seen after transfecting Phox2b alone was reduced by around 60% (Fig. 4F). Similarly, Id2, when co-transfected with Phox2b, consistently prevented the increase in NeuroM+ cells caused by Phox2b; the effect on Delta1 expression was more variable, but on most sections, Delta1 expression was not increased in response to Phox2b (Fig. 4I-J').
Finally, we tested the idea that downregulation of chick Hes5b and Id2 in response to Phox2b may be a consequence of increased Ngn2 expression. Forced expression of Ngn2, however, resulted in a marked upregulation of the chick Hes5b and Id2 expression levels (Fig. 4N-N''). Although induction of chick Hes5b probably reflects activation of the Notch pathway, the mechanism by which Ngn2 promotes Id gene expression remains undefined.
Together, these results show that, in addition to activating proneural
genes, Phox2b inhibits expression of negative regulators of neuronal
differentiation by a separate pathway. They also raise the possibility that
downregulation of Hes5 and Id2 may be essential components
of the response to Phox2b. As Hes5 expression can be taken as a
read-out for Notch activity (de la Pompa
et al., 1997; Ohtsuka et al.,
1999
), Phox2b may affect expression of any of the components
involved in this signaling pathway.
Regulatory interactions between Phox2b and transcription factors
expressed in the progenitor domain
We then explored the transcriptional regulations by which Phox2b specifies
a bm/vm phenotype. There is now a large body of evidence to suggest that, in
addition to driving generic neuronal differentiation, the bHLH factors with
proneural activity play key roles in the specification of neuronal subtype
identity (Perez et al., 1999;
Fode et al., 2000
;
Mizuguchi et al., 2001
;
Novitch et al., 2001
;
Scardigli et al., 2001
;
Lo et al., 2002
;
Parras et al., 2002
). We
therefore tested whether the activation of a bm/vm phenotype in response to
Phox2b could be mediated by Ngn2 or Mash1. Overexpression of Ngn2,
although promoting premature neurogenesis, did not induce bm/vm markers (not
shown). Misexpression of Mash1 resulted in the appearance of ectopic
Islet1+, Islet2- cells in the dorsolateral spinal cord
(Fig. 5A-A''). However,
neither ectopic expression of the motoneuronal marker ChAT
(Fig. 5A''')
nor of Phox2a (not shown) could be detected, two genes that are
induced by Phox2b (Dubreuil et
al., 2000
). In some embryos, we observed ectopic expression of
chicken Phox2b in the transfected area, but this effect was slight
and inconsistent. We thus conclude that neither Ngn2 nor Mash1 mediates the
effect of Phox2b on bm/vm differentiation.
The current model of neuronal subtype specification in the spinal cord
posits that the VZ is parcellated into different domains, each expressing a
different set of transcription factors that cross-repress each other and
ensure that only the appropriate type of neurons arises from each domain
(Briscoe et al., 2000;
Muhr et al., 2001
;
Novitch et al., 2001
;
Lee and Pfaff, 2001
). Changing
the transcriptional code of the progenitor domains may thus be a prerequisite
for inducing ectopic bm/vm neurons. In the ventral hindbrain, the expression
patterns of Phox2b and Nkx2.2 are co-extensive
(Pattyn et al., 2000
), raising
the possibility that Nkx2.2 may be involved in the fate-specifying activity of
Phox2b. However, Phox2b misexpression did not yield ectopic
Nkx2.2+ cells (Fig.
5B,B').
We then tested whether Phox2b affects the expression of
Nkx6.1, Nkx6.2, Pax6 and Olig2. Among them, Nkx6.1
and Nkx6.2 are expressed by hindbrain bm/vm progenitors
(Qiu et al., 1998;
Briscoe et al., 1999
;
Cai et al., 1999
) and are
required for their proper development (J. Ericson and M. Sander, personal
communication), albeit the Nkx6.1/6.2 expression domains also encompasses the
progenitors of sm and V2 neurons. In both, mouse and chick, Nkx6.2 is
co-expressed with Nkx6.1 in bm/vm progenitors, while in the spinal
cord, the expression patterns of both genes are co-extensive in chick but not
in mouse embryos (Cai et al.,
1999
; Vallstedt et al.,
2001
). The ventral limits of Pax6 and Olig2
expression define the dorsal boundary of the bm/vm progenitor domain
(Ericson et al., 1997
;
Osumi et al., 1997
;
Mizuguchi et al., 2001
). In
the absence of Pax6, hindbrain sm neurons appear to acquire a vm
identity (Ericson et al.,
1997
; Osumi et al.,
1997
). Nkx6.1 and Nkx6.2 expression should thus favor a
bm/vm fate, whereas Pax6 and Olig2 should suppress it.
Forced expression of Phox2b resulted in dorsal expansion of the
domains of Nkx6.1 and Nkx6.2 expression whereas
Pax6 and Olig2 were repressed
(Fig. 5C-D''). These data
show that Phox2b changes the pattern of transcription factor
expression in the progenitor domains and suggest that to be able to initiate
bm/vm differentiation, Phox2b needs to downregulate progenitor
factors that are not permissive for this fate. They also suggest that Phox2b
may cooperate with Nkx6.1 and Nkx6.2 in specifying a bm/vm identity.
An activator form of Phox2b mimics the auto-regulatory and
neurogenesis-promoting activities of Phox2b and the induction of Islet1
Only activating functions have so far been ascribed to Phox2b
(Yang et al., 1998;
Lo et al., 1999
;
Yokoyama et al., 1999
;
Flora et al., 2001
). To
investigate whether Phox2b functions as an activator when promoting
neurogenesis and bm/vm differentiation, we fused the Phox2a HD either to the
trans-activating domain of the viral protein VP16 (PHDVP16) or to the
repressor domain of Drosophila Engrailed (PHDEnR). The
Phox2a HD could be used in place of the Phox2b HD, as the two HDs are
identical at the amino acid level (Pattyn
et al., 1997
). In a previous study, the PHDEnR construct
has been shown to behave as a dominant-negative form of Phox2a/b in neural
crest cells (Lo et al., 1999
).
Similar fusions between the HD of Siamois, which is also a member of
the paired homeogene family
(Galliot et al., 1999
), and
the activator or repressor domains were used as controls (SHDVP16 and
SHDEnR).
Expression of PHDVP16 mimicked the ability of Phox2b to elicit emigration to the ML and ectopic Islet1/2+ cells, albeit the effect on Islet1/2 expression was less pronounced. By contrast, expression of PHDEnR had no such effects (Fig. 6A-B'). Similarly, PHDVP16 expression, but not that of PHDEnR or SHDVP16, increased the number of Ngn2+ cells within the transfected area at 6 h.a.e., when the effect of Phox2b on Ngn2 expression was greatest (Fig. 6C,C',E-F'). Ngn1 expression was not yet upregulated at 6 h.a.e. (not shown), but was so at 20 h.a.e. (Fig. 6D,D'). None of these effects was observed after expression of the HD alone (not shown). Positive autoregulation of Phox2b and upregulation of Nkx6.1 also appeared to reflect an activator function, as PHDVP16 transfection resulted in induction of the endogenous Phox2b gene and in dorsal expansion of Nkx6.1 expression (Fig. 6G-H'), whereas PHDEnR had no effect on Phox2b and repressed Nkx6.1 (Fig. 6I-J'). Together, these results suggest that Phox2b acts as a transcriptional activator in promoting neurogenesis and a bm/vm fate.
|
Activation of the endogenous Phox2b gene potentially complicates
the interpretation of the PHDVP16 phenotype, which could be ascribed
to wild-type Phox2b. However, conversion of a transcriptional
repressor to an activator, or the converse, generates a dominant-negative or
antimorphic form that antagonizes the endogenous protein
(Onichtchouk et al., 1998). In
principle, then, if wild-type Phox2b acted as a repressor, PHDVP16 should
block and not phenocopy its activity.
In contrast to the foregoing results, transfection of both PHDVP16
and PHDEnR (but not of SHDVP16) reduced chick Hes5b
and Id2 expression, an effect also observed after expression of the
HD alone (Fig. 7). These
results suggest that inhibition of Hes and Id genes by full-length Phox2b may
be mediated by the HD, and that the function of the Phox2 HD is dominant over
heterologous repressor and activator regions. Equivalent results have been
obtained for the HD transcription factor Xdbx
(Gershon et al., 2000). Also
in this case, the isolated HD and activator and repressor fusion constructs
mimicked the repressive activity of the full-length protein. One possibility
is that the Phox2 HD binds to and sequesters essential co-activators required
for chick Hes5b and Id2 expression. A similar mechanism has
been shown to operate in the case of the inhibition of astrocyte
differentiation by Ngn1 (Sun et al.,
2001
). Likewise, the Pou domain transcription factor Pit1 has been
found to function as a DNA-binding dependent activator and a DNA
binding-independent repressor (Scully and
Rosenfeld, 2002
). Clearly, further work is required to elucidate
the mechanism by which Phox2b represses the expression of these negative
regulators of neurogenesis.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Phox2b upregulates proneural genes and represses negative regulators
of neurogenesis
One function of Phox2b, whether misexpressed in the spinal cord or in its
normal expression domain in the ventral hindbrain, is to increase the
probability of cell cycle exit and to activate pan-neuronal markers
(Dubreuil et al., 2000). Our
present data strongly suggest that this activity is mediated in part by
proneural bHLH factors. Phox2b misexpression expanded the expression
domain of Ngn2, which has been shown to promote generic neuronal
differentiation in a wide variety of settings, including the chick neural tube
(Mizuguchi et al., 2001
;
Novitch et al., 2001
).
Moreover, in combination with Nkx2.2 it induced Cash1, which
is normally co-expressed with Phox2b in bm/vm progenitors, is
downregulated in the absence of Phox2b
(Pattyn et al., 2000
) and
(like Ngn2) capable of driving neuronal differentiation in the chick
neural tube. Another result supporting the view that up-regulation of
proneural genes is required for premature neurogenesis in response to
Phox2b is that co-expression of Id2, a natural inhibitor of
bHLH factor activity, greatly attenuates the effect of Phox2b on
generic neuronal differentiation. There is now ample evidence suggesting that
Mash1 and Ngn2, in addition to their proneural function,
participate in neuronal subtype specification
(Fode et al., 2000
;
Scardigli et al., 2001
;
Lo et al., 2002
). The fact
that Phox2b directs bm/vm differentiation, whether in combination
with Mash1 in its endogenous expression domain or with Ngn2
after forced expression in the spinal cord, suggests that all that Ngn2 and
Mash1 provide in this context is their proneural function.
Phox2b not only upregulates expression of the Ngn2 or
Mash1 proneural genes, but also represses inhibitors of neurogenesis
such as Hes or Id family members
(Ishibashi et al., 1995;
Lyden et al., 1999
;
Ohtsuka et al., 1999
;
Cai et al., 2000
;
Cau et al., 2000
). As
overexpressing Ngn2 increased rather than decreased chick
Hes5b and Id2 expression, downregulation of these genes
cannot be a consequence of Ngn2 induction and probably occurs by a
separate pathway. Several lines of evidence support the idea that
downregulation of chick Hes5b and Id2 plays a causal role in
the neurogenesis-promoting function of Phox2b. Forced expression of
Phox2b results in rapid repression of chick Hesb5 and
Id2, independently of cell cycle stage, suggesting that their
downregulation precedes the last S phase. Furthermore, constitutive expression
of either gene counteracts the premature production of neurons caused by
Phox2b misexpression.
In the Phox2b knockout mice, Hes5 expression is
downregulated rather than stimulated
(Dubreuil et al., 2000) and
there is no change in the expression of Id family members (data not shown).
Downregulation of Mash1 and Delta and the ensuing reduced
activity of the Notch pathway most probably accounts for reduced Hes5
expression. In the light of our evidence that Ngn2 stimulates
Id2 expression, reduced proneural activity may also explain why
Id expression is unchanged in the absence of Phox2b. Hence, a
sustained lack of Phox2b activity as occurs in knock out mice may
induce feedback loops that prevent upregulation of Hes or Id genes and this
may explain why some Phox2b-/- progenitors are still able
to exit the cell cycle.
Finally, the question should be asked why forced expression of
Ngn2 or Mash1 are able to promote neuronal differentiation
in the presence of endogenous chick Hes5b and Id2. First, Hes5, which
downregulates expression of proneural genes in addition to counteracting their
activity (de la Pompa et al.,
1997; Kondo and Raff,
2000
; Ohtsuka et al.,
1999
), cannot repress expression of the transfected genes driven
by artificial promoters. Second, high-level expression of the transgenes as
achieved by transfection may titrate out the repressor molecules. Hence,
forced expression of the proneural bHLH factors may be sufficient to overcome
the influence of these negative regulators of neurogenesis, while
counteracting them by transcription factors such as Phox2b may be required in
a physiological setting.
Phox2b changes the pattern of transcription factor expression in the
progenitor domain
In the ventral hindbrain, Phox2b is expressed both by cycling
bm/vm progenitors and by their postmitotic descendants. Our data argue that
during its neuroepithelial phase of expression, Phox2b acts not only
to promote cell cycle exit but also as a patterning gene, controlling the
identity and fate of dividing progenitors. Pax6 and Olig2
expression defines the dorsal limit of the Nkx2.2+,
Phox2b+ bm/vm progenitor domain
(Ericson et al., 1997;
Briscoe et al., 1999
;
Mizuguchi et al., 2001
). In
the absence of Pax6, spinal interneurons and motoneurons do not properly
develop, and in the caudal hindbrain, sm neurons appear to switch to a vm fate
(Ericson et al., 1997
). Olig2
directs a spinal motoneuronal fate and elicits expression of downstream
factors appropriate for this fate
(Mizuguchi et al., 2001
;
Novitch et al., 2001
).
According to the derepression model of spinal cord neurogenesis
(Muhr et al., 2001
;
Lee and Pfaff, 2001
), both
factors should thus suppress a bm/vm fate. The pronounced downregulation of
the two factors by Phox2b misexpression may thus be a necessary step
in the chain of events that result in the ectopic induction of bm/vm markers.
Pax6 and Phox2b appear to maintain cross-inhibitory
interactions because, in the absence of Pax6, the Phox2b expression
domain in the ventral hindbrain expands dorsally
(Mizuguchi et al., 2001
). One
consequence of Phox2b overexpression is premature neuronal
differentiation, which alone might explain the decrease in progenitor factor
expression. We argue against this explanation, as Phox2b does not
repress Ngn2 and synergizes with Nkx2.2 to activate
Cash1, two genes that in the spinal cord are restricted to progenitor
domains. Because Olig2 expression depends on Pax6
(Mizuguchi et al., 2001
;
Novitch et al., 2001
), its
downregulation may be a consequence of decreased Pax6 expression, but
may also occur by a separate pathway.
Nkx6.1 and Nkx6.2 are co-expressed with Phox2b
in the bm/vm progenitors (Qiu et al.,
1998) and are required for their proper development (J. Ericson
and M. Sander, personal communication). In line with this, Phox2b
misexpression results in dorsal expansion of their expression domains. These
HD factors may thus cooperate with Phox2b in the implementation of a bm/vm
phenotype both in the ventral hindbrain and after misexpression in the spinal
cord. However, Nkx6.1 is expressed throughout the ventral third of
the neural tube and, when ectopically expressed, directs sm and V2 neuronal
fates (Briscoe et al., 2000
).
This shows that by itself, Nkx6.1 does not induce a bm/vm fate and
that Phox2b activity is required to achieve this.
Phox2b acts as an activator in inducing Ngn2 and cranial motoneuronal
markers
The available evidence suggests that Phox2b functions as a transcriptional
activator rather than as a repressor, in line with the fact that it lacks an
EH1 domain that confers repressor activity to other HD proteins
(Muhr et al., 2001). First,
Phox2b binds to and transactivates the promoters of the dopamine
ß-hydroxylase and Phox2a genes
(Yang et al., 1998
;
Yokoyama et al., 1999
;
Adachi et al., 2000
;
Hong et al., 2001
;
Flora et al., 2001
). Second,
the PHDEnR construct, which should act as a repressor, prevents
induction of tyrosine hydroxylase and dopamine ß-hydroxylase by BMP2 in
neural crest cells (Lo et al.,
1999
). We show that PHDVP16 but not PHDEnR
mimics the ectopic induction of Ngn2 and Islet1 in the chick spinal
cord, suggesting that Phox2b functions as an activator.
Phox2b thus appears to provide an activator function, which is necessary
and sufficient for the initiation of bm/vm differentiation. According to the
derepression model of neuronal cell type specification in the spinal cord
(Muhr et al., 2001;
Vallstedt et al., 2001
), the
factors that activate expression of downstream determinants of neuronal
identity are thought to be expressed in the neural tube in a topologically
unrestricted manner. Our results suggest that such transcriptional activators
can also be deployed in spatially restricted domains.
Functional redundancy among neural HD genes
When misexpressed in spinal regions, Phox2b affects the expression
of Pax6, Olig2, Nkx6.1 and Nkx6.2. Nevertheless, expression
of these four genes is not detectably affected in the hindbrain of
Phox2b mutant mice (data not shown), suggesting that other genes are
redundant with Phox2b in this capacity. In the case of Pax6
and Olig2, Nkx2.2, the expression of which does not depend on
Phox2b and which in GOF experiments represses both genes
(Muhr et al., 2001;
Novitch et al., 2001
), is
probably sufficient to exclude Pax6 and Olig2 from bm/vm
progenitors in the absence of Phox2b. Conversely, despite the fact that
Nkx2.2 has the capacity to repress Pax6, lack of Nkx2.2 does
not lead to ventral expansion of the Pax6 domain
(Briscoe et al., 1999
). In the
hindbrain, this may be due to functional redundancy with Nkx2.9, but
on the basis of our results this may also be due to the presence of Phox2b.
Likewise, bm/vm neurons appear to develop normally in the absence of Nkx2.2
(Briscoe et al., 1999
), which
again may be attributed to the persistent expression of either Nkx2.9
or Phox2b.
It has been argued that redundant roles have been selected as backup
against developmental error, whereby one of the genes fails to be expressed
adequately at the appropriate site, thus helping to maintain the
spatiotemporal precision of embryonic patterning
(Cooke et al., 1997). Not
surprisingly, the complex process of neuronal patterning appears to provide
striking examples of this. The alternative possibility is that the redundant
roles we observe are selected because they are non-redundant at other
expression sites.
Synchronizing neuronal fate determination with timing and extent of
neurogenesis
Neurogenesis involves the parallel activation of a program that controls
commitment to cell cycle exit and generic neuronal differentiation and of a
program that specifies the identity of the neurons to be generated. Recent
studies suggest that one way these two programs are coordinated is by the
type-specification properties of bHLH genes themselves. Examples are the bHLH
transcription factors Math1 and Ngn1, the ectopic expression of which in the
neural tube both drives the cells to move to the ML and fosters production of
distinct classes of interneurons (Gowan et
al., 2001), and Mash1, the misexpression of which in the dorsal
forebrain and ventral spinal cord promotes the appearance of ventral cell
types (Fode et al., 2000
) and
V2 interneurons (Parras et al.,
2002
), respectively. Another example is provided by Olig2
expressed in spinal motoneuron progenitors
(Mizuguchi et al., 2001
;
Novitch et al., 2001
). When
misexpressed in the spinal cord or hindbrain, it drives cells to exit the cell
cycle and to migrate to the ML and, in addition, directs expression of
determinants of sm or V2 neuronal fates. Furthermore, Olig2
misexpression expands the domain of Ngn2 expression suggesting that
Olig2, like Phox2b, promotes generic neurogenesis by boosting the expression
of downstream proneural genes. Hence, Olig2 and Phox2b may have comparable
roles in sm and bm/vm progenitors, respectively. However, one important
mechanistic difference between Phox2b and Olig2 is that Phox2b acts as an
activator in inducing Ngn2 and motoneuronal markers, whereas Olig2
functions as a transcriptional repressor. (Whether Olig2 also represses
negative regulators of neuronal differentiation has not been examined.) It
will be interesting to learn which transcription factors play this role in
other neuronal lineages and how they work.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adachi, M., Browne, D. and Lewis, E. J. (2000). Paired-like homeodomain proteins Phox2a/Arix and Phox2b/NBPhox have similar genetic organization and independently regulate dopamine beta-hydroxylase gene transcription. DNA Cell Biol. 19,539 -554.[CrossRef][Medline]
Bertrand, N., Castro, D. S. and Guillemot, F. (2002). Proneural genes and the specification of neural cell types. Nat. Rev. Neurosci. 3, 517-530.[CrossRef][Medline]
Blader, P., Fischer, N., Gradwohl, G., Guillemot, F. and
Strähle, U. (1997). The activity of Neurogenin1 is
controlled by local cues in the zebrafish embryo.
Development 124,4557
-4569.
Briscoe, J. and Ericson, J. (2001). Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11,43 -49.[CrossRef][Medline]
Briscoe, J., Sussel, L., Serup, P., Hartigan-O'Connor, D., Jessell, T. M., Rubenstein, J. L. and Ericson, J. (1999). Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398,622 -627.[CrossRef][Medline]
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101,435 -445.[Medline]
Brunet, J.-F. and Ghysen, A. (1999). Deconstructing cell determination: proneural genes and neuronal identity. Bio Essays 21,313 -318.[CrossRef][Medline]
Cai, J., Amand, T., Yin, H. L., Guo, H. H., Li, G. Y., Zhang, Y. D., Chen, Y. P., Beachy, P. A. and Qiu, M. (1999). Expression and regulation of the chicken Nkx6.2 homeobox gene suggest its possible involvement in ventral neural patterning and cell fate specification. Dev. Dyn. 216,459 -468.[CrossRef]
Cai, L., Morrow, E. M. and Cepko, C. L. (2000).
Misexpression of basic helix-loop-helix genes in the murine cerebral cortex
affects cell fate choices and neuronal survival.
Development 127,3021
-3030.
Castella, P., Sawai, S., Nakao, K., Wagner, J. A. and Caudy,
M. (2000). Hes-1 repression of differentiation and
proliferation in PC12 cells: role for the helix3-helix4 domain in
transcriptional repression. Mol. Cell. Biol.
20,6170
-6183.
Cau, E., Gradwohl, G., Fode, C. and Guillemot, F.
(1997). Mash1 activates a cascade of bHLH regulators in olfactory
neuron progenitors. Development
124,1611
-1621.
Cau, E., Gradwohl, G., Casarosa, S., Kageyama, R. and Guillemot,
F. (2000). Hes genes regulate sequential stages of
neurogenesis in the olfactory epithelium. Development
127,2323
-2332.
Cepko, C. L. (1999). The roles of intrinsic and extrinsic cues and bHLH genes in the determination of retinal cell fates. Curr. Opin. Neurobiol. 9, 37-46.[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]
Cooke, J., Nowak, M. A., Boerlijst, M. and Maynard-Smith, J. (1997). Evolutionary origins and maintenance of redundant gene expression during metazoan development. Trends Genet. 13,360 -363.[CrossRef][Medline]
de la Pompa, J. L., Wakeham, A., Correia, K. M., Samper, E.,
Brown, S., Aguilera, R. J., Nakano, T., Honjo, T., Mak, T. W., Rossant, J. and
Conlon, R. A. (1997). Conservation of the Notch signalling
pathway in mammalian neurogenesis. Development
124,1139
-1148.
Dubreuil, V., Hirsch, M.-R., Pattyn, A., Brunet, J.-F. and
Goridis, C. (2000). The Phox2b transcription factor
coordinately regulates neuronal cell cycle exit and identity.
Development 127,5191
-5201.
Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90,169 -180.[Medline]
Flora, A., Lucchetti, H., Benfante, R., Goridis, C., Clementi,
F. and Fornasari, D. (2001). SP proteins and PHOX2B regulate
the expression of the human PHOX2a gene. J. Neurosci.
21,7037
-7045.
Fode, C., Ma, Q., Casarosa, S., Ang, S. L., Anderson, D. J. and
Guillemot, F. (2000). A role for neural determination genes
in specifying the dorsoventral identity of telencephalic neurons.
Genes Dev. 14,67
-80.
Galliot, B., de Vargas, C. and Miller, D. (1999). Evolution of homeobox genes: Q50 Paired-like genes founded the Paired class. Dev. Genes Evol. 209,186 -197.[CrossRef][Medline]
Gershon, A. A., Rudnick, J., Kalam, L. and Zimmermann, K.
(2000). The homeodomain-containing gene Xdbx inhibits neuronal
differentiation in the developing embryo. Development
127,2945
-2954.
Gowan, K., Helms, A. W., Hunsaker, T. L., Collisson, T., Ebert, P. J., Odom, R. and Johnson, J. E. (2001). Crossinhibitory activities of Ngn1 and Math1 allow specification of distinct dorsal interneurons. Neuron 31,219 -232.[Medline]
Guillemot, F. (1999). Vertebrate bHLH genes and the determination of neuronal fates. Exp. Cell Res. 253,357 -364.[CrossRef][Medline]
Hartigan, D. J. and Rubenstein, J. L. R. (1996). The cDNA sequence of murine Nkx 2.2. Gene 168,271 -272.[CrossRef][Medline]
Henrique, D., Hirsinger, E., Adam, J., Roux, I. L., Pourquie, O., Ish-Horowicz, D. and Lewis, J. (1997). Maintenance of neuroepithelial progenitor cells by delta-notch signalling in the embryonic chick retina. Curr. Biol. 7, 661-670.[Medline]
Hirsch, M. R., Tiveron, M.-C., Guillemot, F., Brunet, J.-F. and
Goridis, C. (1998). Control of noradrenergic differentiation
and Phox2a expression by MASH1 in the central and peripheral nervous system.
Development 125,599
-608.
Hong, S. J., Kim, C.-H. and Kim, K.-S. (2001). Structural and functional characterization of the 5' upstream promoter of the human Phox2a gene: possible direct transactivation by transcription factor Phox2b. J. Neurochem. 79, 1-13.[CrossRef][Medline]
Ishibashi, M., Ang, S.-L., Shiota, K., Nakanishi, S., Kageyama, M. and Guillemot, F. (1995). Targeted disruption of mammalian hairy and Enhancer of split homolog-1 (HES-1) leads to up-regulation of neural helixloop-helix factors, premature neurogenesis, and severe neural tube closure. Genes Dev. 9,3136 -3148.[Abstract]
Jasoni, C. L., Walker, M. B., Morris, M. D. and Reh, T. A.
(1994). A chicken achaete-scute homolog (CASH-1) is expressed in
a temporally and spatially discrete manner in the developing nervous system.
Development 120,769
-783.
Jen, Y., Manova, K. and Benezra, R. (1997). Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis. Dev. Dyn. 208,92 -106.[CrossRef][Medline]
Jessell, T. M. (2000). Neuronal specification in the spinal cord:inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Jouve, C., Palmeirim, I., Henrique, D., Beckers, J., Gossler,
A., Ish-Horowicz, D. and Pourquié, O. (2000). Notch
signalling is required for cyclic expression of the hairy-like gene HES1 in
the presomitic mesoderm. Development
127,1421
-1429.
Kabos, P., Kabosova, A. and Neuman, T. (2002).
Blocking HES1 expression initiates GABAergic differentiation and induces the
expression of p21CIP1/WAF1 in human neural stem cells. J. Biol.
Chem. 277,8763
-8766.
Kageyama, R. and Nakanishi, S. (1997). Helix-loop-helix factors in growth and differentiation of the vertebrate nervous system. Curr. Opin. Genet. Dev. 7, 659-665.[CrossRef][Medline]
Kageyama, R. and Ohtsuka, T. (1999). The Notch-Hes pathway in mammalian neural development. Cell Res. 9,179 -188.[Medline]
Kondo, T. and Raff, M. (2000). Basic
helix-loop-helix proteins and the timing of oligodendrocyte differentiation.
Development 127,2989
-2998.
Koshiba-Takeuchi, K., Takeuchi, J. K., Matsumoto, K., Momose,
T., Uno, K., Hoepker, V., Ogura, K., Takahashi, N., Nakamura, H., Yasuda, K.
and Ogura, T. (2000). Tbx5 and the retinotectum projection.
Science 287,134
-137.
Koyano-Nakagawa, N., Wettstein, D. and Kintner, C. (1999). Activation of Xenopus genes required for lateral inhibition and neuronal differentiation during primary neurogenesis. Mol. Cell. Neurosci. 14,327 -339.[CrossRef][Medline]
Lee, S.-K. and Pfaff, S. L. (2001). Transcriptional networks regulating neuronal identity in the developing spinal cord. Nat. Neurosci. Suppl. 4, 1183-1190.
Lemaire, P., Darras, S., Caillol, D. and Kodjabachian, L. (1998). A role for the vegetally expressed Xenopus gene Mix.1 in endoderm formation and in the restriction to mesoderm to the marginal zone. Development 125,2371 -2380.
Lo, L., Morin, X., Brunet, J.-F. and Anderson, D. J. (1999). Specification of neurotransmitter identity by Phox2 proteins in neural crest stem cells. Neuron 22,693 -705.
Lo, L., Dormand, E., Greenwood, A. and Anderson, D. J. (2002). Comparison of the generic neuronal differentiation and neuron subtype specification functions of mammalian achaete-scute and atonal homologs in cultured neural progenitor cells. Development 129,1553 -1567.
Lu, Q. R., Sun, T., Zhu, Z., Ma, N., Garcia, M., Stiles, C. D. and Rowitch, D. H. (2002). Common developmental requirement for Olig function indicates a motor neuron/ oligodendrocyte connection. Cell 109,75 -86.
Lyden, D., Young, A. Z., Zagzag, D., Yan, W., Gerald, W., O'Reilly, R., Bader, B. L., Hynes, R. O., Zhuang, Y., Manova, K. and Benezra, R. (1999). Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401,670 -677.
Ma, Q., Kintner, C. and Anderson, D. J. (1996). Identification of neurogenin, a vertebrate neuronal determination gene. Cell 87,43 -52.
Martinsen, B. J. and Bronner-Fraser, M. (1998). Neural crest specification regulated by the helix-loop-helix repressor Id2. Science 281,988 -991.
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.
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K. and Yasuda, K. (1999). Efficient targeting of gene expression in chick embryos by microelectroporation. Dev. Growth Diff. 41,335 -344.
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.
Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J. (1996). A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogenesis. Dev. Biol. 174,233 -247.
Nakamura, Y., Sakakibara, S., Miyata, T., Ogawa, M., Shimazaki, T., Weiss, S., Kageyama, R. and Okano, H. (2000). The bHLH gene Hes1 as a repressor of the neuronal commitment of CNS stem cells. J. Neurosci. 20,283 -293.
Norton, J. D. (2000). ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J. Cell Sci. 113,3897 -3905.
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.
Ohnuma, S.-i., Philpott, A. and Harris, W. A. (2001). Cell cycle and cell fate in the nervous system. Curr. Opin. Neurobiol. 11, 66-73.
Ohtsuka, T., Ishibashi, M., Gradwohl, G., Nakanishi, S., Guillemot, F. and Kageyama, R. (1999). Hes1 and Hes5 as Notch effectors in mammalian neuronal differentiation. EMBO J. 18,2196 -2207.
Onichtchouk, D., Glinka, A. and Niehrs, C. (1998). Requirement for Xvent-1 and Xvent-2 gene function in dorso-ventral patterning of Xenopus mesoderm. Development 125,1447 -1456.
Osumi, N., Hirota, A., Ohuchi, H., Nakafuku, M., Iimura, T., Kuratani, S., Fujiwara, M., Noji, S. and Eto, K. (1997). Pax-6 is involved in the specification of hindbrain motor neuron subtype. Development 124,2961 -2972.
Parras, C. M., Schuurmans, C., Scardigli, R., Kim, J., Anderson, D. J. and Guillemot, F. (2002). Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity. Genes Dev. 16,324 -338.
Pattyn, A., Morin, X., Cremer, H., Goridis, C. and Brunet, J.-F. (1997). Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 124,4065 -4075.
Pattyn, A., Morin, X., Cremer, H., Goridis, C. and Brunet, J.-F. (1999). The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399,366 -370.
Pattyn, A., Hirsch, M.-R., Goridis, C. and Brunet, J.-F. (2000). Control of hindbrain motor neuron differentiation by the homeobox gene Phox2b. Development 127,1349 -1358.
Perez, S. E., Rebelo, S. and Anderson, D. J. (1999). Early specification of sensory neuron fate revealed by expression and function of neurogenins in the chick embryo. Development 126,1715 -1728.
Qiu, M., Shimamura, K., Sussel, L., Chen, S. and Rubenstein, J. L. R. (1998). Control of anteroposterior and dorsoventral domains of Nkx-6.1 gene expression relative to other Nkx genes during vertebrate CNS development. Mech. Dev. 72, 77-88.
Roztocil, T., Matter-Sadzinski, L., Alliod, C., Ballivet, M. and Matter, J. M. (1997). NeuroM, a neural helix-loop-helix transcription factor, defines a new transition stage in neurogenesis. Development 124,3263 -3272.
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.
Scully, K. M. and Rosenfeld, M. G. (2002). Pituitary development: regulatory codes in mammalian organogenesis. Science 295,2231 -2235.
Sechrist, J. and Marcelle, C. (1996). Cell division and differentiation in avian embryos: techniques for study of early neurogenesis and myogenesis. In Methods in Cell Biology, Vol. 51 (ed. M. Bronner-Fraser), pp. 5-15. San Diego, CA: Academic Press.
Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z, Zubiaga, A., Hua, X., Fan, G. and Greenberg, M. E. (2001). Neurogenin promotes neurogenesis and inhibits glia differentiation by independent mechanisms. Cell 104,365 -376.
Takebayashi, K., Akazawa, C., Nakanishi, S. and Kageyama, R. (1995). Structure and promoter analysis of the gene encoding the mouse helix-loop-helix factor HES-5. J. Biol. Chem. 270,1342 -1349.
Toma, J. G., El-Bizri, H., Barnabé-Heider, F., Aloyz, R. and Miller, F. D. (2000). Evidence that helix-loop-helix proteins collaborate with retino-blastoma tumor suppressor protein to regulate cortical neurogenesis. J. Neurosci. 20,7648 -7655.
Triezenberg, S. J., Kingsbury, R. C. and McKnight, S. L. (1988). Functional dissection of VP16, the transactivator of herpes simplex virus immediate early gene expression. Genes Dev. 2,718 -729.
Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell, T. M. and Pfaff, S. L. (1994). Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79,957 -970.
Vallstedt, A., Muhr, J., Pattyn, A., Pierani, A., Mendelsohn, M., Sander, M., Jessell, T. M. and Ericson, J. (2001). Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 31,743 -755.
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.
Yang, C., Kim, H.-S., Seo, H., Kim, C.-H., Brunet, J.-F. and Kim, K-S. (1998). Paired-like homeodomain proteins, Phox2a and Phox2b, are responsible for noradrenergic cell-specific transcription of the dopamine beta-hydroxylase gene. J. Neurochem. 71,1813 -1826.
Yokoyama, M., Watanabe, H. and Nakamura, M. (1999). Genomic structure and functional characterization of NBPhox (PMX2B), a homeodomain protein specific to catecholaminergic cells that is involved in second messenger-mediated transcriptional activation. Genomics 59,40 -50.
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.