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First published online 30 April 2008
doi: 10.1242/dev.015115
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Division of Molecular Neurobiology, National Institute of Medical Research, The Ridgeway, Mill Hill, NW7 1AA London, UK.
* Author for correspondence (e-mail: fguille{at}nimr.mrc.ac.uk)
Accepted 3 April 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: bHLH, Proneural, Dentate gyrus, Hippocampus, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Altman and Das,
1965; Kempermann et al.,
1997a; Kempermann et al.,
1997b). Neurogenesis in the adult DG has been observed in a
variety of species, including primates and humans
(Eriksson et al., 1998;
Gould et al., 1999a;
Gould et al., 1998;
Kempermann and Gage, 1998;
Kuhn et al., 1996). The DG is
the primary afferent pathway into the hippocampus and it has an important role
in learning and memory. Maintenance of neurogenesis in the adult DG has been
suggested to play a role in the acquisition of new memories
(Aimone et al., 2006;
Gould et al., 1999b;
Lemaire et al., 2000).
Formation of the DG begins in mice at around E15 in the dorsomedial part of
the telencephalic vesicles. The DG primordium is initially populated by
Cajal-Retzius cells and radial glial cells that are likely to participate to
its histogenesis (Alcantara et al.,
1998; Borrell et al.,
1999; Del Rio et al.,
1997; Rickmann et al.,
1987). The portion of hippocampal neuroepithelium that constitutes
the DG primordium, also called primary matrix, contains stem/progenitor cells
that give rise, starting at E15.5, to a stream of migratory progenitors and
postmitotic neurons that has been called the secondary matrix. At the end of
their migration, progenitor cells of the secondary matrix accumulate in the
tertiary matrix, located in the hilar region of the hippocampus, where they
give rise, from 
E17 onwards, to the granule neurons of the DG, which are
organized in a V-shaped laminar structure
(Altman and Bayer, 1990;
Cowan et al., 1980). Although
the secondary matrix starts to disappear by P5, progenitors from the tertiary
matrix persist throughout life in a region located at the interface between
the hilus of the hippocampus and the granular cell layer, termed the
subgranular layer (SGL) (Altman and Bayer,
1990; Altman and Das,
1965). These progenitors produce throughout adulthood new granule
neurons that have the same electrophysiological properties than neurons
generated during embryonic and early postnatal development
(Laplagne et al., 2006).
Whether adult hippocampal stem cells reside in the SGL of the DG
(Ming and Song, 2005) or near
the lateral ventricle of the HP (Seaberg
and van der Kooy, 2002) is a matter for debate.
The molecules that control the development of the DG and particularly
determine cell fates in this structure remain poorly characterized. A range of
defects in formation of the hippocampus has been observed in mice in which the
Wnt signalling pathway is disrupted. Wnt3a mutants present a deletion
of the whole hippocampus (Lee et al.,
2000), while Lef1 mutants lack most of the DG
(Galceran et al., 2000) and
LRP6 mutants have a reduced number of DG progenitors and granule
neurons (Zhou et al., 2004).
Wnt signalling acts in part by promoting expression of the homeodomain protein
Emx2, which is required for growth of the hippocampus and for migration of DG
progenitors (Backman et al.,
2005; Oldekamp et al.,
2004; Pellegrini et al.,
1996; Theil et al.,
2002; Tole et al.,
2000). Transcription factors of the basic helix-loop-helix (bHLH)
class, including Neurod1 and NEX/Math2 (Neurod6 - Mouse Genome Informatics),
are mainly expressed in postmitotic granule cells and have been implicated in
late stages of differentiation of dentate granule neurons
(Liu et al., 2000;
Miyata et al., 1999;
Pleasure et al., 2000;
Schwab et al., 2000). By
contrast, little is known of the transcription factors regulating early stages
of neurogenesis in the DG and particularly the generation and initial
differentiation of the different populations of progenitors involved in
development of the DG.
Proneural bHLH proteins control the generation of progenitor cells and
their progression through the neurogenic programme throughout the nervous
system (Bertrand et al., 2002).
Expression of the proneural protein Mash1 (Ascl1 - Mouse Genome Informatics)
has been reported in DG progenitors at embryonic and postnatal stages
(Pleasure et al., 2000), but
its role in formation of the DG has not been assessed. The proneural protein
neurogenin 2 (Ngn2) plays an essential role in neurogenesis in the dorsal
telencephalon, where it has been shown to commit multipotent progenitors to
the neuronal fate and inhibit astrocytic differentiation. Ngn2 also activates
a cortical-specific differentiation programme that includes expression of
transcription factors such as Neurod1 and NEX/Math2, resulting in acquisition
of a glutamatergic neurotransmission phenotype and pyramidal neuronal
morphology (Hand et al., 2005;
Nieto et al., 2001;
Schuurmans et al., 2004).
Ngn2 is expressed in the developing DG
(Pleasure et al., 2000) but
its function in development of this structure has not yet been addressed.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mice
were genotyped as described by Seibt et al.
(Seibt et al., 2003) and
Casarosa et al. (Casarosa et al.,
1999). Both lines were maintained in an outbred MF1 background.
Wild-type, heterozygous and homozygous Ngn2KIGFP mutant
mice were obtained from intercrosses of Ngn2GFP/+ mice.
The morning of the day on which the vaginal plug was observed was termed E0.5;
the day of birth was termed P0. Mash1::GFP mice where identified at
birth by GFP expression under a UV microscope. All experiments were carried
out in accordance with the UK (Scientific Procedures) Animal Act 1986.
RNA in situ hybridization
Embryonic and postnatal brains were dissected out of the skull and fixed at
4°C in paraformaldehyde (4%) overnight. Brains were then rinsed in
phosphate-buffered saline (PBS), cryoprotected overnight in 20% sucrose in
PBS, embedded in OCT (BDH, UK), and sectioned on a cryostat at 10 µm. RNA
in situ hybridization was performed as described by Cau et al.
(Cau et al., 1997). The RNA
probes used in this study were the following: Ngn1, Ngn2, Ngn3, Mash1
and Neurod1 (Cau et al.,
1997); Prox1 (Torii
et al., 1999).
Immunohistochemistry
Brains were dissected as mentioned above and fixed at 4°C in
paraformaldehyde (4%) for 30 minutes then cut thought the midline in half and
fixed in the same solution for another 30 minutes. Antigen retrieval for Ki67
antibody staining was performed by heating sections in PBS at 65°C for 5
minutes. Sections were incubated in a blocking solution (PBS plus 10% normal
goat serum (Vector Laboratories) and 0.1% Tween20 or Triton X-100) and then
with primary antibodies overnight at 4°C. The following primary antibodies
were used: mouse monoclonal antibodies anti-GFAP (1/500, Sigma), IgG2b
anti-HuC/D (1/200, Molecular Probes), IgG1 anti-Mash1 (1/10; a gift from D. J.
Anderson), IgG2a anti-Ngn2 (1/20; a gift from D. J. Anderson); rat monoclonal
antibodies IgG2a anti-BrdU (1/20, Oxford Biotechnology), anti-Ki67 (1/50,
Novocastra) and anti-PDGFR
(1/800, DB biosciences); rabbit antisera
anti-caspase 3 activated (1/1000, R&D Systems), anti-GFP (1/1000,
Molecular Probes), anti-phosphohistone H3 (1/1000, Upstate), anti-Olig2
(1/1000, Chemicon) and anti-Prox1 (1/3000, Covance Research Products); goat
anti-Neurod1 (1/100, Santa Cruz Biotechnology); and chicken anti-GFP (1/500,
Chemicon). Corresponding secondary antibodies were incubated for 1 hour at
room temperature, including Alexa Fluor 568-conjugated goat (or donkey)
anti-mouse, anti-rabbit, anti-rat or anti-goat; and Alexa Fluor 488 goat (or
donkey)-conjugated anti-rabbit, anti-mouse or anti-chicken (all from Molecular
Probes, 1/1000). DAPI (1/5000) was used to label DNA and sections were mounted
in Aquapolymount medium (Polysciences). Images were captured using SP1 and SP2
confocal microscopes (Leica, Germany), Radiance 2100 (BioRad, UK) confocal
microscope and Zeiss Imager Z1 (Zeiss, Germany) with the Apotome system.
Histology, BrdU incorporation and TUNEL experiments
For histological analysis, brains were fixed overnight in Bouin's fixative,
processed for wax embedding, cut at 6 µm, and stained with Haematoxylin and
Eosin. For BrdU incorporation experiments, pregnant females or P1 pups were
injected intraperitoneally with 100 µg/g of body weigh of BrdU (Sigma) and
sacrificed after 30 minutes. For immunohistochemistry, sections were processed
as described above and BrdU incorporation was exposed by 30 minutes treatment
in HCl 2N at 37°C. The TUNEL experiment was carried out following the
supplier manual (ApopTag Kit, Qbiogene).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Pleasure et al., 2000),
Ngn2 transcripts were detected in these three cell populations
(Fig. 1A). Similarly, Ngn2
protein could be detected in a subset of cells in the three DG matrices
(Fig. 1B). The numbers of cells
expressing Ngn2 decreased from the primary to the secondary matrix and from
the secondary to the tertiary matrix (Fig.
1C), in parallel with the progressive reduction in the proportion
of progenitor cells present in these populations
(Altman and Bayer, 1990).
In the developing neocortex, Ngn2 is expressed in dividing
progenitors and is rapidly downregulated as cells leave the cell cycle
(Britz et al., 2006;
Gradwohl et al., 1996;
Hand et al., 2005). To
determine whether this is also the case in the DG, we examined Ngn2 expression
in dividing progenitors marked by a 30-minute pulse of BrdU or by Ki67
(Key et al., 1993), and in
post-mitotic granule neurons marked by expression of the homeobox
transcription factor Prox1 (Oliver et al.,
1993; Liu et al.,
2000). We found that a fraction of Ngn2+ cells
incorporated BrdU in the three DG matrices (12%, 43% and 38% respectively;
Fig. 1D-D''') and a
majority of them expressed Ki67 (e.g. 85% in the tertiary matrix;
Fig. 1G), indicating that they
correspond mainly to progenitor cells. By contrast, only a small fraction of
Ki67+ or BrdU+ progenitor cells expressed Ngn2 in any of
the three matrices (Fig.
1D-D''',G). To determine whether Ngn2 expression is
maintained in post-mitotic granule neurons, we examined the co-expression of
Ngn2 and Prox1. We detected two cell populations expressing Prox1 at markedly
different levels in the DG (see Fig. S1 in the supplementary material). Most
cells of the primary matrix and a large fraction of cells in the secondary
matrix, but fewer cells were in the tertiary matrix and the dentate gyrus
itself, expressed Prox1 at a low level (Prox1low)
(Fig. 1E-E''). Most
Prox1low cells (82%) expressed the dividing cell marker Ki67,
indicating that they are progenitors (see Fig. S2 in the supplementary
material). By contrast, cells expressing Prox1 at a high level
(Prox1high) were absent from the primary matrix and sparse in the
secondary matrix but constituted the main cell population of the dentate
gyrus. These cells were Ki67 negative and therefore correspond to post-mitotic
granule neurons (see Fig. S2 in the supplementary material). Accordingly, Ngn2
was expressed only by Prox1low and not by Prox1high
cells (Fig. 1E-E''),
indicating that Ngn2 expression is downregulated when progenitors exit the
cell cycle and is not maintained in post-mitotic granule neurons.
Expression of the bHLH protein Neurod1 is restricted in the DG to
post-mitotic neurons (see Fig. S3B in the supplementary material), although
Neurod1 transcripts are also found in BrdU-incorporating progenitors
(see Fig. S3A in the supplementary material)
(Lee et al., 2000). Double
labelling for Ngn2 and Neurod1 showed that the two proteins are expressed in
non-overlapping cell populations, thus confirming that Ngn2 expression in the
developing DG is confined to mitotic progenitors
(Fig. 1F-F'').
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). GFP
expression in Ngn2KIGFP heterozygous mice recapitulates
Ngn2 expression, and as the GFP protein is maintained in the recent progeny of
Ngn2-expressing progenitors because of its greater stability, it can be used
to trace the short-term fate of these progenitors
(Fig. 2A-C)
(Britz et al., 2006).
Injection of BrdU in Ngn2KIGFP newborn mice 30 minutes
before analysis revealed that most BrdU+ progenitors (80%)
express GFP (Fig. 2D-D''),
indicating that they had previously expressed Ngn2. Thus, Ngn2 is transiently
expressed by most DG progenitors.
To confirm that Ngn2-expressing progenitors give rise to dentate granule
neurons, we examined the expression of the granule neuron markers Prox1 and
Neurod1, and the general neuronal marker HuC/D
(Wakamatsu and Weston, 1997)
in Ngn2KIGFP mice. Over 90% of GFP+ cells
expressed Prox1 in all three matrices, with an increasing fraction of cells
displaying the high expression levels found in dentate neurons, as they
progress from the primary to the tertiary matrix
(Fig. 2E-E''). Forty to
50% of GFP+ cells also expressed HuC/D in the secondary and
tertiary matrix (Fig.
2F-F'') and a similar fraction expressed Neurod1
(Fig. 2G-G''). These data
indicate that Ngn2-expressing progenitors give rise to postmitotic
Prox1high, Neurod1+, HuC/D+ dentate granule
neurons. Over 90% of Prox1+ cells expressed GFP, thus confirming
that most DG neurons originate from Ngn2-expressing progenitors.
We also examined Ngn2 expression and GFP expression in Ngn2KIGFP mice at the onset of DG development (E16.5, Fig. 3). Ngn2-expressing cells were abundant in the primary matrix and some were also found to contribute to the emerging secondary matrix. A significant fraction of these cells incorporated BrdU after a 30-minute pulse (11% in the primary matrix and 30% in the secondary matrix; Fig. 3A) and almost all of them expressed Ki67, indicating that they are progenitors (Fig. 3B). Moreover, all Prox1high granule neurons already expressed GFP in Ngn2KIGFP mice at this early stage (Fig. 3C). Thus, Ngn2 is already expressed in dentate granule neuron progenitors at the beginning of DG development.
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Reduced generation of dentate gyrus progenitors in Ngn2 mutant mice
We then examined whether the DG phenotype in
Ngn2KIGFP/GFP mutant brains was due to a defect in the
generation or in the survival of dentate granule neurons. At E15.5 and E16.5,
the number of progenitors labelled by BrdU after a 30 minutes pulse was
reduced by 20-25% in the primary and secondary matrices of Ngn2
mutant mice when compared with wild types
(Fig. 5A,A',C). At E18.5,
the number of progenitors was reduced by 30% in the primary and secondary
matrices and by 65% in the tertiary matrix
(Fig. 5B,B',C). The
number of GFP+ cells (i.e. derived from Ngn2+
progenitors) was also reduced in Ngn2KIGFP/GFP embryos, as
the fraction of GFP+ that had incorporated BrdU was similar in
Ngn2KIGFP/+ and Ngn2KIGFP/GFP embryos
(between 20 and 30%; Fig. 5D).
Ki67 labelling also revealed a reduction in number of cycling cells in
Ngn2KIGFP/GFP at E16.5 (24% in the secondary matrix,
Fig. 5A,A',E) and at
E18.5 (20%, 26% and 48% in the primary, secondary and tertiary matrix,
respectively, Fig.
5B,B',E). The ratio of cells in S-phase (BrdU+) over
dividing cells (Ki67+) was similar in Ngn2 mutant and wild-type mice,
suggesting that the number of dividing progenitors rather than their cell
cycle length is affected by the loss of Ngn2. The number of
Prox1low-expressing progenitors was also reduced in Ngn2
mutant embryos at E16.5 (Fig.
6A,A',C). Activated caspase 3
(Fig. 6D) and TUNEL (data not
shown) labelling revealed a small but not significant increase in apoptosis in
Ngn2 mutant DG at E15.5, E16.5 and E18.5
(Fig. 6D and not shown). This
suggests that the reduction in progenitor cell number in Ngn2 mutant
DG reflects mainly a defect in the generation of progenitors, although a
reduced ability to survive may play a minor role in this phenotype.
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). We thus asked whether Mash1 is also involved in DG
development and particularly if it contributes to the generation and/or
differentiation of the remaining DG progenitors in Ngn2 mutant mice.
We first compared the expression of Mash1 in the developing DG with that of
Ngn2. At P1, a large fraction of Ngn2+ progenitors co-expressed
Mash1 in the tertiary matrix (63±5% of Ngn2+ cells are
Mash1+ and 53±8% of Mash1+ cells are
Ngn2+; Fig. 8A,A1).
A majority of Mash1-expressing cells (64±7%) co-expressed
Proxlow, suggesting that, like Ngn2+ cells, these cells
are dentate granule progenitors (Fig.
8B,B1). In a Mash1::GFP reporter line
(Gong et al., 2003), we found
that a larger fraction of Ngn2+ progenitors co-expressed GFP
(74±6%) than Mash1 protein (61±5%), suggesting that some
Ngn2+ progenitors had expressed and then downregulated Mash1
(Fig. 8C). Similarly, in
Ngn2KIGFP/+ mice, a larger fraction of Mash1+
cells co-expressed GFP than Ngn2 (67±5% and 51±4%;
Fig. 8A,D), suggesting that
some Mash1+ cells had expressed and then downregulated Ngn2.
The extensive co-expression of Mash1 and Ngn2 in DG progenitors suggested
that the two factors might share some functions in DG neurogenesis, as
previously reported in other parts of the telencephalon
(Nieto et al., 2001). We
therefore examined whether Mash1 is required during development of
the DG and/or whether it compensates for the loss of Ngn2 in the DG
progenitors that remain in Ngn2 mutants. The expression of
Neurod1 and Prox1 (Fig.
8E-H), and the number of progenitors labelled by phophohistone H3
and Ki67 (see Fig. S5A,A' in the supplementary material) were not
affected in Mash1 mutant embryos at E18.5, indicating that
Mash1 function is not essential for the formation of the DG at
prenatal stages. Mash1 function at postnatal stages could not be
assessed owing to the death of Mash1 mutants at birth. To determine
whether Mash1 can compensate for the loss of Ngn2 during DG
development, we examined Ngn2; Mash1 double mutant mice.
Prox1high- and Prox1low-expressing cells were similarly
reduced in the DG of Ngn2 single mutants, and Ngn2, Mash1
double mutant embryos at E18.5 (Fig.
8F''-H; see Fig. S4 in the supplementary material),
suggesting that Mash1 cannot compensate for the loss of Ngn2
during the prenatal phase of DG development.
In addition to the activation of neurogenesis, Mash1 has been
shown to inhibit astrogliogenesis (Nieto
et al., 2001) and to promote oligodendroglial development
(Parras et al., 2004;
Parras et al., 2007;
Battiste et al., 2007;
Sugimori et al., 2007;
Sugimori et al., 2008).
However, expression of the astrocyte marker GFAP and of the oligodendrocyte
precursor markers PDGFR and Olig2 was not changed in the hippocampus of
Mash1 mutants at birth (see Fig. S5 and Fig. S6A,A' in the
supplementary material). Olig2 expression was also unchanged in Ngn2
mutant and Ngn2, Mash1 double mutant embryos (see Fig.
S6A'',A''' in the supplementary material).
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).
Math3 appears to be expressed in DG progenitors (see Fig. S7B in the
supplementary material), while Math2 expression is confined to
dentate granule neurons (Pleasure et al.,
2000). Math3 expression persists in the DG progenitors
that remain in Ngn2 mutant embryos (see Fig. S7B' in the
supplementary material), suggesting that it may promote neurogenesis to some
extent in Ngn2 mutant embryos.
Cells accumulate at the periphery of the Ngn2 mutant DG
Analysis of GFP expression in the DG of both
Ngn2KIGFP/GFP and Ngn2KIGFP/+ mice
revealed the presence of GFP+ cells at the periphery of the upper
blade of the DG (Fig.
9A,A',B,B',E). Double labelling experiments for GFP
and progenitor markers (Ngn2+, BrdU+,
Prox1low; Fig.
9C-C'1,F,G) or neuronal differentiation markers
(Hu+, Neurod1+; Fig.
9D,D',G) showed that these peripheral cells include both
progenitors and post-mitotic neurons (Fig.
9F,G). This peripheral cell population seemed to be more packed in
mutant than wild-type mice, particularly after birth
(Fig. 9B,B'), and it
presented a differentiation defect similar to that observed in the secondary
matrix, with progenitors (i.e. Ngn2+, BrdU+,
Prox1low cells) being present in larger numbers in Ngn2
mutant than in wild-type mice (Fig.
9G). These mutant cells may fail to migrate inwardly, from the
periphery of the DG into the granule cell layer, resulting in their relative
accumulation in this peripheral location
(Fig. 9E; see Discussion).
Radial glial cells, which express the astrocytic marker GFAP in the DG,
have been implicated in the outward migration of newborn neurons from the
subgranular layer to the granule cell layer
(Rickmann et al., 1987). Thick
bundles of GFAP+ radial glial fibres were found at the periphery of
the wild-type DG, and these fibres crossed the lower and upper blades of the
DG with an orientation perpendicular to the long axis of the blades. The
GFAP+ bundles were still present at the periphery of the reduced
Ngn2 mutant DG, but no fibres were found crossing the DG blades
(Fig. 9C-C'1). This lack
of radial glial fibres may perturb the migration of DG neurons, resulting in
their accumulation at the periphery of the DG in Ngn2 mutants at
birth and in the abnormal distribution of GFP+ cells observed in
the Ngn2 mutant DG at later stages (see Fig. S8 in the supplementary
material).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ngn2 expression in dentate gyrus progenitors
Nng2 protein is transiently expressed by progenitors of the DG, identified
by their proliferative state and by low level of expression of the homeodomain
protein Prox1. Ngn2 expression is then downregulated before progenitors become
post-mitotic and differentiate, as marked by the expression of Neurod1, HuC/D
and Prox1 at high level. Using a Ngn2KIGFP reporter mouse,
we have traced the fate of Ngn2+ cells and shown that they give
rise to most dentate granule neurons.
Another proneural protein, Mash1, has also been found in progenitor cells
of the developing and adult DG (Pleasure
et al., 2000). We show here that Ngn2 and Mash1 are expressed in
the same DG progenitor lineage and that the two factors are largely
co-expressed in DG progenitors, suggesting that they are both involved in DG
neurogenesis. Another bHLH protein Neurod1, is also present in the developing
DG but its expression is restricted to postmitotic, differentiating neurons
(Pleasure et al., 2000).
Neurod1+ cells are absent from the primary matrix, the portion of
embryonic neuroepithelium from which all DG progenitors originate, but
Neurod1+ cells are found intermingled with Ngn2+
progenitors in the secondary matrix, indicating that this migratory cell
population is heterogeneous and contains cells at different stages of
maturation along the dentate granule neuron lineage. The postmitotic
Neurod1+ neurons found in the secondary matrix might be born in the
primary matrix (Pleasure et al.,
2000) or in the secondary matrix from migratory progenitors that
become postmitotic and begin to differentiate while migrating.
The functions of Ngn2 in the developing dentate gyrus
In Ngn2 mutant mice, there is a strong reduction in size of the
forming DG at the end of embryonic development (E18.5). Mutant mice that
escape perinatal lethality have an almost complete loss of the lower blade of
the DG and a reduced upper blade (Fig.
4). This is the first report of a proneural factor being required
in DG progenitors for normal DG development.
Ngn2 mutant mice present a marked reduction in number of dividing progenitors in all matrices of the DG without a major increase in cell death, suggesting that Ngn2 function is required for the generation and expansion of DG progenitors. Loss of Ngn2 does not appear to affect the duration of the cell cycle of progenitors, as the fraction of cells in S-phase (BrdU+) among all dividing progenitors (Ki67+) remains the same in Ngn2 mutant and wild-type mice. Thus, loss of Ngn2 may result in a cell cycle arrest of DG progenitors that would normally continue to proliferate. In addition, Ngn2 mutant progenitors do not differentiate properly, as shown by the reduced fraction of Ngn2+ progenitor-derived cells expressing the neuronal markers HuC/D and βIII-tubulin. It is presently unclear whether this is due to a delay or to a complete block in expression of these markers. Unexpectedly, Neurod1 appears to be normally expressed by Ngn2 mutant DG neurons, suggesting that Ngn2 regulates only some aspects of the differentiation programme of dentate granule cells.
The proneural protein Mash1 is also expressed by DG progenitors
(Pleasure et al., 2000)
(Fig. 8), raising the
possibility that it regulates aspects of the dentate granule neuron phenotype
not controlled by Ngn2, or that it takes over some of the functions of
Ngn2 when this gene is mutated. However, analysis of Mash1
single mutants and Mash1; Ngn2 double mutants does not
support a significant role for Mash1 in DG neurogenesis in a
wild-type or Ngn2 mutant context up to birth, when these mice die.
Other neurogenin genes (Ngn1 and Ngn3) are not detectably
expressed in the developing DG, but the bHLH gene Math3/Neurod4 is
expressed in both wild-type and, at a reduced level, Ngn2 mutant DG,
suggesting that it may partially compensate for the absence of Ngn2
and drive DG neurogenesis in Ngn2 mutants. Alternatively, another yet
unidentified factor that may not belong to the bHLH transcription factor
family (e.g. Jafar-Nejad et al.,
2006) may share some of Ngn2 activities, including the regulation
of Neurod1 and be involved in DG development along with Ngn2.
Ngn2 has been shown to specify several aspects of the subtype
identity of projection neurons in the cerebral cortex, including their
glutamatergic neurotransmission phenotype and their pyramidal morphology
(Hand et al., 2005;
Schuurmans et al., 2004).
Interestingly, although DG granule cells also originate from Ngn2-expressing
progenitors, they have very different characteristics from cortical projection
neurons, and in particular present a mixed glutamatergic and GABAergic
phenotype (Gutierrez, 2003;
Gutierrez, 2005), and a
granule cell morphology very distinct from that of pyramidal cortical neurons.
Although there is no overt defect in expression of glutamatergic and GABAergic
markers in the DG of Ngn2 mutant embryos (see Fig. S9 in the
supplementary material), it is tempting to speculate that both Ngn2 [a
glutamatergic neuron determinant
(Schuurmans et al., 2004)] and
Mash1 [a GABAergic neuron determinant
(Fode et al., 2000)] may
contribute to the specification of the unique identity of DG granule cells.
Testing this hypothesis will require to examine the phenotype of DG granule
cells at postnatal stages in conditional Mash1 and Ngn2
mutant mice.
A new route for the migration of DG progenitors?
We have found progenitor cells located in the external part of the dentate
granular layer in the developing DG. Altman and Bayer
(Altman and Bayer, 1990)
previously described the presence of proliferating cells in this outer region
and assumed that these were glial progenitors, but our molecular analysis
identifies them instead as dentate granule cell progenitors (BrdU+,
Ngn2KIGFP+ and Prox1low). This suggests that, at least
at embryonic stages, a fraction of granule neuron progenitors could reach the
DG by migrating along the outer border of the lower and upper blades of the
DG, rather than along the inner side of the lower blade towards the tertiary
matrix, as usually assumed. Once located at the periphery of the DG, the outer
progenitors presumably produce granule neurons (some of them found in a
peripheral position, Fig. 9)
that may reach their final location in the granule cell layer via an inward
migration route. Testing this model will require tracing the migration of DG
progenitors and granule neurons by timelapse imaging. Moreover,
GFP+ cells expressing either progenitor (Prox1low, BrdU
or Ki67) or postmitotic neuronal markers (Prox1high, Neurod1 or
HuC/D) appear to accumulate in Ngn2 mutant mice at the periphery of
the remaining DG blade, suggesting that mutant neurons may fail to migrate
inwards and into the granule cell layer. If a migration defect indeed takes
place in the Ngn2 mutant DG, the disruption of the radial glia
scaffold revealed by GFAP staining may be involved in this phenotype.
Although the mechanism by which loss of Ngn2 results in disruption
of radial glial cells is unclear, it is noteworthy that a similar phenotype
has been observed in mice mutant for the Wnt co-receptor Lrp6
(Zhou et al., 2004). The
similarity in phenotype between Lrp6 and Ngn2 mutant mice
extends to a reduction in number of dentate granule neurons. Ngn2 has
been shown to be directly regulated by Wnt signalling
(Hirabayashi et al., 2004;
Israsena et al., 2004),
suggesting that it may mediate some of the functions of the Wnt signalling
pathway in DG development. Wnt signalling has also been implicated in the
regulation of neurogenesis in the adult DG
(Lie et al., 2005), and
Ngn2 expression is maintained in progenitor cells in the subgranular
layer of the postnatal DG (Ozen et al.,
2007), thus raising the exciting possibility that a Wnt
signalling-Ngn2 pathway controls both the development of the DG and the
maintenance of neurogenesis in the adult structure.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/2031/DC1
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Laboratoire de Biologie des Interactions Neurones/Glie
INSERM U-711, Université Pierre et Marie Curie, IFR des Neurosciences,
Hôpital de la Salpetrière, 75651 Paris Cedex 13, France 
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0.05, **P
