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First published online 4 July 2007
doi: 10.1242/dev.02870
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KAN Research Institute Inc., KobeMI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
Author for correspondence (e-mail:
y-ono{at}kan.eisai.co.jp)
Accepted 22 May 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Helt, Neurogenin, Basic-helix-loop-helix, Transcriptional repressor, GABAergic neurons, Glutamatergic neurons, Mesencephalon, Neuronal identity, Transmitter phenotype determination
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Jessell,
2000; Schuurmans and
Guillemot, 2002). The subtype identity, which is determined by the
expression of selective sets of transcription factors, defines the
neurotransmitter usage, migration and axonal innervation pattern.
GABAergic and glutamatergic neurons are the principal inhibitory and
excitatory neurons in the brain, respectively. One of the best-characterized
regions involved in GABAergic versus glutamatergic transmitter decision
control is the developing dorsal spinal cord. In this region, homeodomain and
basic helix-loop-helix (bHLH) transcription factors play pivotal roles in the
specification of the progenitors and derived neurons
(Cheng et al., 2004;
Cheng et al., 2005;
Glasgow et al., 2005;
Gross et al., 2002;
Mizuguchi et al., 2006;
Muller et al., 2002). Among
these postmitotically expressed transcription factors, Ptf1a and
Tlx1/3 function as selector genes that switch alternative transmitter
fates (Cheng et al., 2004;
Cheng et al., 2005;
Glasgow et al., 2005). By
contrast, in the telencephalon, the proneural bHLH factors Mash1 and Ngn2
(also known as Ascl1 and Neurog2, respectively - Mouse Genome Informatics) are
selectively expressed in progenitors for GABAergic and glutamatergic neurons,
respectively, and are involved in the determination of transmitter phenotype
(Fode et al., 2000;
Parras et al., 2002),
suggesting that the transmitter is chosen at the progenitor state. Proneural
factors are also involved in the specification of dorsal spinal interneurons
(Gowan et al., 2001;
Helms et al., 2005;
Muller et al., 2005;
Nakada et al., 2004). However,
the concept that transmitter selection is simply controlled by proneural genes
at the progenitor state, as in the telencephalon, is unlikely in the dorsal
spinal cord (Mizuguchi et al.,
2006; Wildner et al.,
2006). Thus, acquisition of the GABAergic or glutamatergic
phenotype is controlled by distinct pathways in different brain areas. The
mechanisms of transmitter selection have not yet been elucidated in other
brain regions, such as the mesencephalon.
Previous studies have described arcuate expression patterns of
transcription factors that might specify the neuronal subtypes in the
mesencephalon (Agarwala and Ragsdale,
2002; Sanders et al.,
2002). However, the transmitter patterns have not been accorded
with the neuronal subtypes defined by transcription factor codes. Thus, the
neuronal identity, location of neuron emergence and control of GABAergic and
glutamatergic neuron development in the mesencephalon remain largely
unknown.
The bHLH-Orange (bHLH-O) family consists of members related to Hes proteins
that show conserved primary structures and transcriptional repressor functions
(Davis and Turner, 2001). In
neuronal development, Hes genes act as downstream effectors for the Notch
pathway and inhibit neuronal differentiation by repressing proneural bHLH
factor expression (Bertrand et al.,
2002; Kageyama,
1999; Ross et al.,
2003). This inhibitory function of Hes genes is required for
lateral inhibition of neurogenesis, which controls progenitor maintenance and
the timing of neuronal birth, and local organizer development in the neural
tube (Baek et al., 2006;
Bertrand et al., 2002;
Kageyama, 1999;
Ross et al., 2003). However,
whether bHLH-O factors are involved in neuronal subtype specification remains
uncertain.
Recently, we and others identified a novel bHLH-O family member,
Helt (also known as Heslike and Megane), which is
selectively expressed in neural progenitors in the developing mesencephalon
and diencephalon (Guimera et al.,
2006a; Miyoshi et al.,
2004; Nakatani et al.,
2004). Gain- and loss-of-function studies revealed that
Helt has a potent activity for promoting mesencephalic GABAergic
neuron development (Guimera et al.,
2006b; Miyoshi et al.,
2004). However, it has not been addressed whether Helt is
only involved in the promotion of GABAergic neuron differentiation or whether
it also plays a role in transmitter phenotype selection. Furthermore,
downstream target genes of Helt have not yet been identified, and thus the
mechanism of action of Helt remains to be clarified.
In the present study, we found that Helt functions as a selector gene that determines the GABAergic over glutamatergic transmitter phenotype in the mesencephalon. Furthermore, we identified Ngn (Neurog) genes, which are selectively expressed in glutamatergic progenitors in the mesencephalon and show activity for promoting glutamatergic differentiation, as downstream target genes of Helt. By using a mesencephalic domain map delineated by transcription factor expression, we further found that the Helt and Ngn pathways are commonly used in mesencephalic GABAergic and glutamatergic differentiation and that transmitter choice in mesencephalic neurons is not completely coupled with the specification of neuronal subtype, which might be regulated by homeodomain transcription factors. Taken together, our results illustrate the strategy of the transmitter phenotype decision in mesencephalic neuron development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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pNE was constructed by ligating the SV40 poly(A) signal and genomic
fragments for the intronic enhancer and promoter of the nestin gene
(Miyoshi et al., 2004) into
pSP73 (Promega). pNE-Helt was constructed by ligating a Helt cDNA
(Nakatani et al., 2004) into
the MluI/NotI sites of pNE. pNE-Ngn1 was constructed by
ligating an Ngn1 cDNA into the KpnI/NotI sites of
pNE. The primer sequences used for amplification of these fragments are
available upon request. Linearized pNE constructs were injected into
fertilized eggs and founder embryos were collected at E12.5. The embryos were
genotyped by PCR.
Immunohistochemistry and in situ hybridization
Immunohistochemistry was performed as described previously
(Nakatani et al., 2004). The
polyclonal rabbit anti-Helt antibody was also described previously
(Nakatani et al., 2004). A rat
anti-Nkx6.1 monoclonal antibody was raised against GST-Nkx6.1 (aa 60-122). The
other primary antibodies used were: anti-Lim1/2, anti-Nkx2.2 and anti-Pax7
(Developmental Studies Hybridoma Bank); anti-Mash1 (BD Pharmingen); anti-Lim1,
anti-Ngn1, anti-Ngn2 and anti-Hnf3ß (Santa Cruz Biotechnology); and
anti-Brn3a (Chemicon).
In situ hybridization was performed as described previously
(Nakatani et al., 2004). The
primer sequences used for amplification of probe cDNAs (Gad1, Vglut2
and GFP) are available upon request.
FACS
Ventral mesencephalons were dissected from E12.5 homozygous and
heterozygous Helt mutant embryos and dissociated using Accumax
(Chemicon). Cell sorting was performed using a FACS Aria (BD Biosciences).
Total RNA was isolated from 
5x104 sorted GFP+
cells. cDNA amplicons were prepared as described
(Osada et al., 2005) and the
expression of homeobox and proneural genes was analyzed by RT-PCR (primer
sequences available upon request).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
whereas the adjacent m6 domain produced glutamatergic red nucleus (RN) neurons
(Fig. 1B)
(Fedtsova and Turner, 2001).
Helt was selectively expressed in the m2 to m5 domains at E11.5 and
Gad1+ GABAergic neurons emerged from these domains as
previously reported (Fig. 1A,B)
(Guimera et al., 2006a;
Miyoshi et al., 2004;
Nakatani et al., 2004). The
neurons emerging from the m3 to m5 domains appeared to have distinct
identities despite their common GABAergic phenotype and Helt expression in
their progenitors, as they expressed different sets of postmitotically
expressed transcription factors, such as Nkx2.2 and Hnf3ß, in addition to
the distinct transcription factor codes in their progenitors
(Fig. 1A). The m2 domain,
marked by Nkx2.2 expression in the mantle layer (ML), generated both GABAergic
and glutamatergic neurons in a mosaic fashion
(Fig. 1B). Nkx2.2 appeared to
selectively mark GABAergic neurons in the m2 domain
(Fig. 1B). The dorsal-most m1
domain marked by Pax7 expression only generated glutamatergic neurons at E11.5
(Fig. 1A,B). However, at later
stages (E12.5-13.5), both GABAergic and glutamatergic neurons were generated
in a salt-and-pepper pattern (Fig.
1A). From E11.5 onward, Helt started to be expressed in this
domain as a ventral-to-dorsal wave, and at E12.5 Helt was expressed in all
areas of the domain (Fig. 1A).
Taken together, these observations indicate that GABAergic neurons are
generated from five of the seven mesencephalic progenitor domains, and that
Helt is coincidently and commonly expressed in all the GABAergic progenitor
domains, but is excluded from glutamatergic domains, in the developing
mesencephalon (summarized in Fig.
2).
Helt is required for selection of GABAergic over glutamatergic neuronal fate in the mesencephalon
To examine the role of Helt in GABAergic neuron development, we
generated Helt-null mutant mice by a targeted disruption approach,
and confirmed the null phenotype by the complete loss of Helt expression in
homozygous mutant (Helt-/-) embryos
(Fig. 3A). At E11.5,
Gad1 expression was mostly absent from Helt-/-
mesencephalons, with the exception of some Gad1+ neurons
that emerged from the m5 domain (Fig.
3B). Until at least E13.5, some Gad1+ neurons
were generated in the m3 and m5 domains (see also
Fig. 4B), but the total number
of Gad1+ neurons in the ventral mesencephalon region of
the mutant embryos did not increase to the level found in wild-type control
embryos (Fig. 3B; data not
shown), indicating that a delay in GABAergic neuron generation or
Gad1 induction in postmitotic neurons does not cause this phenotype.
In the dorsal m1 domain, GABAergic neurons were completely lost in the mutants
at E12.5 and E13.5 (Fig. 3B;
data not shown). These results are consistent with recently published
observations that superior colliculus GABAergic neurons derived from the
dorsal mesencephalon are absent in another Helt-knockout mouse strain
(Guimera et al., 2006b). In
addition to this dorsal phenotype, our precise analysis (see also the results
described below) revealed that Helt is required for GABAergic
differentiation in the ventral mesencephalon.
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To determine whether the phenotype was caused by the inability of GABAergic precursors to mature into Gad1+ neurons despite maintaining their correct identity, or by conversion of their fate into other neuronal subtypes, we examined the expression of other transmitter markers, including Vglut2 (also known as Slc17a6 - Mouse Genome Informatics) (Fig. 3B; data not shown). At E11.5, Vglut2 was ectopically expressed at the expense of Gad1 expression in the m3 to m5 domains of the mutant mesencephalons (Fig. 3B). At E12.5, Vglut2 expression in the m1 domain was upregulated in the absence of Helt (Fig. 3B). Thus, Helt activity appears to be required for suppression of glutamatergic neuron generation as well as for induction of GABAergic neurons.
To examine whether Helt is indeed capable of suppressing the
glutamatergic phenotype as well as inducing the GABAergic phenotype, we
generated transgenic mice expressing Helt under the control of the
nestin enhancer. As previously reported using similar transgenic mice
(Miyoshi et al., 2004),
ectopically expressed Helt induced Gad1+ neurons
(Fig. 3C). Importantly, ectopic
Helt expression suppressed the generation of
Vglut2+ neurons (Fig.
3C). Taken together, the results obtained from these loss- and
gain-of-function experiments suggest that Helt plays key roles in the
selection of the GABAergic over glutamatergic transmitter phenotype at the
progenitor state in the mesencephalon.
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),
Helt might be involved in the regional patterning of the neural tube.
To better understand the cause of the Helt mutant phenotype and the
mechanism of action of Helt in controlling the transmitter phenotype
decision, we searched for genes up- or downregulated by the loss of
Helt activity. For this purpose, we compared the gene expression
patterns in presumptive Helt+ progenitors sorted from
Helt+/- and Helt-/- mesencephalons. As
expected from the targeting strategy of replacing the Helt coding
sequence with a GFP cDNA, GFP was transcribed with an
essentially identical pattern to that of Helt, and GFP+
cells could be sorted by FACS (see Fig. S1A,B in the supplementary material).
Among approximately 200 homeodomain factors screened by RT-PCR, none of the
factors selectively expressed in VZ progenitors in the mesencephalon was
identified as showing altered expression after loss of Helt (data not
shown). Consistently, the homeodomain factors expressed within the
Helt+ domain, such as Nkx2.2 and Nkx6.1, were normally expressed in
the VZ of the mutants, and the expression of the VZ transcription factors
Hnf3ß and Sim1 was also unaffected
(Fig. 4A; data not shown). In
addition, as expected from these observations, the expression patterns of
these factors were not affected by ectopic Helt expression in the
transgenic embryos (see Fig. S2 in the supplementary material). These results
suggest that Helt is not involved in progenitor domain formation.
Importantly, postmitotic expression of Nkx2.2, which was selectively expressed
by Gad1+ populations among postmitotic neurons in the
mesencephalon, was not affected in the m4 domain, although Nkx2.2 expression
in the postmitotic neurons in the m2 domain was lost
(Fig. 4A). In addition,
although some Nkx2.2+ neurons retained Gad1 expression in
the Helt-/- mutants at E12.5, most Nkx2.2+
neurons expressed Vglut2, suggesting that the fate of
Nkx2.2+ neurons was converted from GABAergic to glutamatergic in
the absence of Helt, even though their neuronal identity was
maintained, at least in part (Fig.
4B). Thus, Helt appears to select the transmitter phenotype
without completely changing the neuronal subtype identity defined by the
homeodomain factor codes.
Helt controls pan-GABAergic and pan-glutamatergic pathways in postmitotic neurons
We reasoned that the expression of postmitotic transcription factors that
control Gad1 or Vglut2 expression might be changed by the
loss of Helt activity. Owing to the long half-life of GFP proteins,
the sorted GFP+ cells contained postmitotic precursors that
originated from the Helt+ domain (see Fig. S1C in the supplementary
material). Thus, by the above-described RT-PCR screening for homeodomain
factors, we were able to examine the postmitotic events affected by the
Helt mutation. We found that some postmitotically expressed factors
were differentially expressed between Helt+/- and
Helt-/- GFP+ cell populations (data not shown).
Among these, we focused on Lim1 and Brn3a (also known as Lhx1 and Pou4f1,
respectively - Mouse Genome Informatics).
We first examined the normal expression patterns of these factors. At
E11.5, Lim1 was selectively expressed in the m2 to m6 domains
(Fig. 5A; data not shown). At
later stages, Lim1 expression was initiated in some populations in the m1
domain along with Gad1 (Fig.
5A,B). Brn3a expression was detected in the ventral-most region of
the basal plate (m6) and the dorsal m1 and m2 domains
(Fig. 5A). Double staining
revealed coexpression of these factors in the m6 domain, which probably
consists of RN neurons (Agarwala and
Ragsdale, 2002; Fedtsova and
Turner, 2001). In all the other regions, Lim1 and Brn3a showed
mutually exclusive expression patterns. By comparison with Gad1 and
Vglut2 expression, we found that Lim1+ domains, except for
the RN domain, were well matched with Gad1 expression, and confirmed
the coexpression of Lim1 and Gad1 at the single cell level
(Fig. 5B). By contrast, Brn3a
expression was specific for Vglut2+ cells, although
Vglut2 expression was also detected in dopaminergic neurons in the m7
domain. In summary, Brn3a specifically marks glutamatergic neurons, whereas
Lim1+ Brn3a- populations define GABAergic neurons, in
the developing mesencephalon (summarized in
Fig. 2).
Next, we examined whether these homeodomain factors were affected by the loss or gain of Helt activity. In the Helt-null mutant at E11.5, Lim1 expression was mostly absent from the m2 to m4 domains, although Lim1+ neurons were observed within the m5 domain, where Gad1 expression was also partially retained (Fig. 6A). At later stages, Lim1 expression in the m5 domain was maintained and some Lim1+ neurons emerged from the m3 and m4 domains (data not shown), consistent with the above observation that some Nkx2.2+ Gad1+ neurons were detected in the mutants. In the dorsal m1 domain, Lim1 expression was completely lost at E12.5 and E13.5 (Fig. 6A; data not shown). By contrast, ectopic Brn3a expression was observed in the m2 to m4 domains of the mutants, where Lim1 expression was lost, and virtually all the neurons emerging from the m1 domain were Brn3a+ (Fig. 6A). GFP+ neurons were generated and migrated toward the ML in the null mutants and, importantly, 95.82±1.09% of GFP+ neurons in the m1 domain expressed Brn3a (Fig. 6A, n=5), indicating that neurogenesis by presumptive Helt+ progenitors was unaffected, but the derived neurons were transfated to become Brn3a+ glutamatergic neurons following the loss of Helt activity. Consistent with these mutant phenotypes, ectopic expression of Helt induced Lim1+ neurons that coexpressed Gad65 (also known as Gad2 - Mouse Genome Informatics) and inhibited Brn3a+ neurogenesis instead (Fig. 6B). Taken together, these results suggest that Helt functions as a selector gene that promotes the pan-GABAergic pathway involving Lim1 by suppressing the pan-glutamatergic pathway involving Brn3a.
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), we reasoned that factors expressed in the VZ and
controlling glutamatergic fate might be downstream targets of Helt. The fact
that bHLH proneural factors play important roles in neuronal subtype
specification and transmitter selection as well as neurogenesis control in
other brain regions (Fode et al.,
2000; Gowan et al.,
2001; Helms et al.,
2005; Muller et al.,
2005; Nakada et al.,
2004; Parras et al.,
2002), and that bHLH-O factors frequently regulate bHLH genes
(Davis and Turner, 2001), led
us to examine the possibility that Helt might regulate bHLH gene expression to
determine the transmitter phenotype. We screened for bHLH factors expressed in
the VZ that were differentially expressed in GFP+ cells sorted from
Helt+/- and Helt-/- mesencephalons,
and identified the Ngn genes (data not shown).
First, we examined the expression patterns of these proneural factors in
the mesencephalon of wild-type embryos. Ngn1 (also known as Neurog1 - Mouse
Genome Informatics) was selectively expressed in the VZ of the
Brn3a+ glutamatergic m1, m2 and m6 domains
(Fig. 7A,B). Ngn2 was
selectively expressed in the RN domain (m6) and ventral midline dopaminergic
domain (m7) at high levels and in the dorsal m1 domain at a relatively low
level and frequency at E11.5 (Fig.
7A,B) (Fedtsova and Turner,
2001; Kele et al.,
2006). At later stages, Ngn2 expression was increased in the m1
domain (Fig. 7A). Importantly,
Helt and the Ngns showed mutually exclusive patterns in all domains at all
stages (Fig. 7A). By contrast,
Mash1 was expressed in all progenitor domains of the developing mesencephalon
and coexpressed with Helt (Fig.
7A) (Miyoshi et al.,
2004).
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To examine whether Helt indeed represses Ngn gene expression in
Helt+ GABAergic progenitors, we analyzed the Ngn expression
patterns in Helt-deficient embryos. At E11.5, ectopic expression of
both factors was observed in the ventral GFP+ m2 to m4 domains,
where Brn3a was ectopically induced and Lim1 expression was lost in the
mutants (Fig. 7D). At E12.5,
Ngn expression was upregulated in the m1 domain of the mutant embryos. More
importantly, Ngns were expressed in the majority of the GFP+
presumptive Helt-expressing progenitors in the VZ
(Fig. 7D; Ngn1,
57.0±4.3%; Ngn2, 73.6±1.5%). Thus, Ngn expression was
derepressed in the Helt+ progenitors after the loss of
Helt activity. By contrast, Mash1 expression was not affected by the
loss of Helt function, indicating that Helt selectively regulates the
Ngn genes and that the role of Helt in promoting GABAergic differentiation
does not involve maintenance of Mash1 expression, which is essential for
GABAergic differentiation in the ventral mesencephalon
(Miyoshi et al., 2004). Taken
together, these observations demonstrate that Ngns are downstream target genes
of Helt in the developing mesencephalon.
Ngn1 can promote glutamatergic differentiation
The selective Ngn expression in glutamatergic progenitors and its
repression by Helt suggest that the repression of Ngn genes might be a key
function of Helt for suppression of the glutamatergic fate. To
examine whether derepression of Ngns by loss of Helt is a primary
cause of transmitter phenotype switching, we first compared the increase in
the number of Ngn1-expressing progenitors in the VZ at E12.5 with the increase
in glutamatergic neurogenesis, as indicated by the rate of accumulated
glutamatergic neurons in the ML at E13.5, in the m1 domain of Helt
mutants as compared with wild-type controls. In the VZ at E12.5, an
approximately 1.51-fold increase in the percentage of Ngn1+ cells
was observed in the m1 domain of the mutant embryos (wild type,
13.86±2.34%; mutant, 20.89±2.05%; n=5). In the ML at
E13.5, the percentage of Brn3a+ glutamatergic neurons in the m1
domain of the mutant embryos increased from 58.96±2.06% to
93.83±2.07% (n=5), which is consistent with the reduction in
Lim1+ GABAergic neurons from 37.73±1.64% to 0%
(n=5). Thus, glutamatergic generation was concomitantly increased
1.59-fold by the loss of Helt. These results support the idea
that derepression of Ngn1 expression by loss of Helt leads to
acquisition of the glutamatergic fate, at least in the m1 domain.
To examine whether Ngn1 is indeed capable of determining the glutamatergic fate, we generated transgenic mice expressing Ngn1 under the control of the nestin enhancer. In the ventral Helt+ domains of Ngn1-transgenic embryos at E11.5, ectopic Brn3a+ neurons were observed, despite the fact that Helt expression did not appear to be affected (Fig. 8A). We next examined whether Ngn1 could promote glutamatergic differentiation in the m1 domain, where a strong correlation between Ngn1 derepression and increased glutamatergic generation was observed in the Helt-null mutants. However, possibly owing to premature differentiation induced by the proneural activity of Ngn1, transgene-expressing progenitors were rare in the transgenic embryos at E12.5. To mark the neurons derived from exogenous Ngn1-expressing progenitors, we generated transgenic mice expressing Ngn1 and IRES-controlled GFP under the control of the nestin enhancer (NE-Ngn1-GFP). As expected from the long duration of GFP protein expression, GFP+ postmitotic neurons could be detected in the ML of the transgenic embryos (Fig. 8B). Importantly, more than 96% of the GFP+ neurons expressed Brn3a (193/201, n=2), whereas none of them expressed Lim1 (0/201, n=2), indicating that exogenous Ngn1-expressing progenitors only generated glutamatergic neurons. Taken together, these results demonstrate that Ngn1 has the potency to promote glutamatergic differentiation and that repression of Ngns by Helt is a prerequisite for GABAergic differentiation in the mesencephalon.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sanders et al., 2002).
However, the characteristics of each neuronal subtype regarding transmitter
usage and function remain largely elusive. In the present study, we have
delineated the transcription factor codes of the progenitor domains in the
mesencephalon and defined the transmitter phenotype of the derived neurons in
each domain. Importantly, Helt defines all the GABAergic progenitors, whereas
the Ngns, which are repressed by Helt, mark glutamatergic progenitors.
Furthermore, we identified pan-GABAergic and pan-glutamatergic postmitotically
expressed transcription factors (Lim1 and Brn3a, respectively). Definition of
the neuronal subtypes and their progenitor domains will be useful for
understanding the developmental mechanism of the mesencephalon.
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; Miyoshi et al.,
2004). Our present knockout approach revealed a pivotal role for
Helt in the selection of the GABAergic over glutamatergic transmitter
phenotype fate. The phenotype of embryos lacking Helt in the
mesencephalon was similar to that of mutants of another bHLH factor,
Ptf1a, in the cerebellum and dorsal spinal cord, in which GABAergic
neurons were lost and glutamatergic neurons were generated instead
(Glasgow et al., 2005;
Hoshino et al., 2005). Thus,
specification of GABAergic neurons over glutamatergic neurons appears to be
achieved using similar mechanisms with distinct bHLH factors, Helt and Ptf1a,
in each brain region. However, the mechanisms of transcriptional control by
these factors might be different, as Helt is a bHLH-O type transcriptional
repressor and Ptf1a is a bHLH factor that heterodimerizes with E proteins and
has a transcriptional activator function
(Beres et al., 2006;
Miyoshi et al., 2004;
Nakatani et al., 2004). Thus,
the downstream genes regulated by these factors are likely to differ.
Consistent with this notion, the phenotypes of null mutants for Helt
and Ptf1a show important differences. In the Ptf1a mutant,
GABAergic neurons originating from Ptf1a+ progenitors were
completely transfated to become other neuronal populations with the
glutamatergic phenotype in the dorsal spinal cord (i.e. dI4 to dI5, dILA to
dILB) (Glasgow et al., 2005).
By contrast, in the Helt mutant, despite the almost complete
conversion of the transmitter phenotype and the expression of pan-GABA and
pan-glutamatergic transcription factors, the expression of Nkx2.2, which was
specifically expressed in subpopulations of GABAergic neurons in the
mesencephalon, was maintained. These observations suggest that Helt, and also
possibly the Ngns, might select the transmitter phenotype without directly
affecting the expression of transcription factors that determine the neuronal
subtype identity in the mesencephalon. Thus, it is suggested that in the
ventral mesencephalic neurons, the pathways that determine neurotransmitter
identity are not completely linked to the pathways regulating neuronal subtype
identity.
Although we cannot exclude the possibility that Helt is also
involved in subtype identity specification, as other identity markers have not
yet been identified, the maintenance of Nkx2.2 expression in m4 neurons of
Helt mutants suggests that determination of the neuronal transmitter
phenotype is not a downstream event of subtype identity determination, at
least in m4 neurons. This idea is supported by the observation that pathways
common to each transmitter fate (i.e. Helt-Lim1-Gad1 and Ngn-Brn3a-Vglut2)
appear to be used in the differentiation of all mesencephalic GABAergic and
glutamatergic neurons. The question then arises as to whether these types of
determination pathway are commonly used for transmitter choice in all brain
regions. In the dorsal spinal cord, all the known factors with transmitter
selector functions, such as Ptf1a, Lbx1 and Tlx1/3, are also involved in the
control of neuronal identity - the pattern of expression of transcription
factors that define neuronal identity is almost completely changed to one that
directs another fate in the absence of these selector factors
(Cheng et al., 2004;
Cheng et al., 2005;
Glasgow et al., 2005;
Gross et al., 2002;
Muller et al., 2002). By
contrast, loss of Pax2, which is commonly expressed by GABAergic
neurons and required for acquisition of the GABAergic phenotype in the spinal
cord, does not result in conversion to another transmitter phenotype
(Cheng et al., 2004). Thus,
Pax2 is not a selector for the transmitter phenotype, but instead
only appears to be required for the promotion of GABAergic differentiation.
Taken together, at least in the dorsal spinal cord, transmitter phenotype and
neuronal subtype identity appear to be tightly coupled with each other, or the
transmitter phenotype might be determined as a downstream event of the
specification of neuronal subtype identity, in contrast to the situation in
the mesencephalon. In the present study, we did not examine whether the
GABAergic phenotype was changed to the glutamatergic phenotype following
maintenance of neuronal identity in the dorsal mesencephalic regions. Future
detailed analyses are required to clarify whether Helt only plays
important roles in transmitter selection or whether, in some cases, it is also
involved in the selection of neuronal identity from two alternative fates,
similar to Ptf1a in the dorsal spinal cord.
|
;
Kageyama and Ohtsuka, 1999;
Ross et al., 2003;
Sakamoto et al., 2003;
Satow et al., 2001). Here, we
have demonstrated that Helt has a similar activity in proneural gene
regulation, consistent with the structural conservation. However, there are
significant differences between the activity of Helt and those of other bHLH-O
members, in that Helt selectively regulates Ngn1 and Ngn2, whereas other
bHLH-O factors repress all proneural genes. Thus, unlike Hes1 and Hes5, Helt
is not a general regulator of neurogenesis, as loss or gain of Helt function
was consistently found to have no effect on neurogenesis. Therefore, the
question arises as to how Helt regulates Ngn expression. Ngn expression was
affected by the gain and loss of Helt activity without any
alterations in the expression of progenitor transcription factors, such as
homeobox factors, which might be involved in the specification of progenitor
identity. Thus, it is unlikely that the repression or derepression of Ngns is
a secondary consequence of the change in neural progenitor identity.
Consistently, Helt repressed Ngn1 promoter activity in transfection assays
(our unpublished observations), suggesting the likely possibility that Helt
directly represses Ngn expression.
|
; Helms et al.,
2005). Thus, the Helt and Ngn pathways might be capable of
controlling the neurotransmitter phenotype without regulating neurogenesis,
which is controlled by the Mash1 pathway. Together with the established roles
of Ngns in glutamatergic determination in the telencephalon
(Fode et al., 2000;
Parras et al., 2002), their
(1) coincident expression in glutamatergic progenitor domains; (2) the strong
correlation between ectopic induction or suppression of Ngn expression and
glutamatergic neurogenesis in Helt-null mutants and
Helt-transgenic embryos; and (3) the potency of Ngn1 to
promote glutamatergic differentiation revealed by Ngn1
gain-of-function studies, all suggest that repression of the Ngn genes is
likely to be a key role of Helt in transmitter selection and indicate that it
is at least essential for suppressing the glutamatergic pathway.
|
|
). Thus,
the default status of mesencephalic progenitors might be GABAergic as
conferred by the ubiquitous expression of Mash1, and Helt might promote
GABAergic differentiation by repressing the Ngns that promote the
glutamatergic pathway (Fig. 9).
This idea is supported by the observation that some GABAergic neurons were
generated in the ventral domains even in the absence of Helt
(Fig. 3B), and the fact that
the increased GABAergic production in these regions with development
correlated well with Ngn downregulation (data not shown), which is regulated
by an unknown mechanism independent of Helt. Loss-of-function studies
for the Ngn genes will be needed to clarify this point and to gain further
understanding of the overall picture of transmitter phenotype determination in
the mesencephalon.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2783/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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