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First published online 7 March 2007
doi: 10.1242/dev.001255
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1 Division of Developmental Biology, Cincinnati Children's Hospital Research
Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA.
2 Department of Neurobiology, University of Tokyo Graduate School of Medicine,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
3 Division of Molecular Neurobiology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London NW7 1AA, UK.
4 Departments of Pediatrics and Neurosurgery, University of Cincinnati College
of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267, USA.
5 Solution Oriented Research for Science and Technology (SORST), Japan Science
and Technology Agency, 3-4-15, Nihonbashi, Chuo-ku, Tokyo 103-0027,
Japan.
Author for correspondence (e-mail:
masato.nakafuku{at}cchmc.org)
Accepted 31 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Stem cell, Cell fate, Cell lineage, Neuron, Glia, Transcription factor, HLH factor, Homeodomain factor, Spinal cord, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In general,
neurons differentiate at early embryonic stages, whereas two glial lineages
emerge later. Thus, their cell fate specification has often been described as
a binary choice, such as `neuron versus glia'. The actual spatiotemporal
pattern of neuro/gliogenesis, however, is much more complex. Recent studies
have demonstrated that three neural lineages emerge in a specific order in a
topologically defined domain. Moreover, cells in the same lineage often
differentiate at distinct times at different positions
(Ross et al., 2003;
Rowitch, 2004). Thus, the
mechanisms for lineage specification are precisely coordinated with those for
determining the timing and position of their differentiation. Both
cell-intrinsic and -extrinsic mechanisms appear to underlie such coordination.
For instance, many extracellular signals have been implicated in the
transition from early neurogenesis to later gliogenesis
(Panchision and McKay, 2002).
Developmental changes in the intrinsic properties of multipotent progenitors
are also involved in this temporal switch
(Qian et al., 2000). Complex
interactions between extracellular signals and intracellular transcriptional
regulators also underlie the link between the positional identity and
cell-type specificity (Jessell,
2000). How these temporal and spatial control mechanisms are
coordinated remains poorly understood.
One of the key cell-intrinsic regulators for neuro/gliogenesis is the
proneural and inhibitory classes of helix-loop-helix (HLH) transcription
factors (Bertrand et al., 2002;
Kageyama et al., 2005). How
they control the timing and position of differentiation, however, remains
unclear. For instance, although Id and Hes families of inhibitory HLH factors
have been implicated in astrocyte differentiation, their expression occurs
much earlier than the onset of astrogenesis
(Kageyama et al., 2005).
Likewise, Olig2, which is essential for oligodendrocyte development, is also
involved in the generation of motoneurons
(Lu et al., 2002;
Zhou and Anderson, 2002;
Takebayashi et al., 2002).
Thus, the expression of these molecules per se is not directly correlated with
the timing of differentiation of specific cell lineages. Moreover, recent
studies have shown that oligodendrocytes arise from multiple, distinct regions
at different timings. Their differentiation overlaps with early neurogenesis
in some regions, whereas it occurs in parallel to astrogenesis at later stages
in other regions (Soula et al.,
2001; Fu et al.,
2002; Cai et al.,
2005; Vallstedt et al.,
2005; Fogarty et al.,
2005; Kessaris et al.,
2006). How such a stage- and region-specific cell genesis pattern
is controlled remains unknown.
To address this issue, we chose the developing ventral spinal cord as a
model. We found that Pax6, Olig2 and Nkx2.2 (Nkx2-2 - Mouse Genome
Informatics), which have previously been shown to specify the positional
identity of multipotent progenitors
(Shirasaki and Pfaff, 2002),
also play crucial roles in determining the timing of differentiation of
neurons and glia through the modulation of the activities of proneural and
inhibitory HLH factors. Here we propose a model whereby these two classes of
transcription factors coordinately regulate the spatiotemporal pattern of
neuro/gliogenesis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Parras et al., 2002). Embryos
of these mice and wild-type Sprague-Dawley rats were collected from
timed-pregnant females.
Immunostaining and RNA in situ hybridization
Rabbit antibodies against Hes1 and Sox2 were prepared by immunization with
synthetic oligopeptides corresponding to their predicted amino acid sequences.
Antibodies for Nkx6.1 (Nkx6-1) and Ngn3 (Neurog3), Ngn2 (Neurog2), and Irx3
were kind gifts from M. German (University of California, San Francisco, CA),
D. J. Anderson (California Institute of Technology, Pasadena, CA) and K.
Shimamura (Kumamoto University, Japan), respectively. Mouse monoclonal
antibodies against Pax7, Nkx2.2, Isl1 and HB9 were obtained from the
Developmental Studies Hybridoma Bank at Iowa University. The antibodies for
the following antigens were purchased from commercial sources: Ngn1 (Neurog1),
O4 and chondroitin sulfate proteoglycan 4 (NG2; Cspg4), Millipore; Mash1 and
Id2, Pharmingen; Id1, Santa Cruz Biotechnology; ß-tubulin type III
(TuJ1), Babco; glial fibrillary acidic protein (GFAP), S100ß, and
2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNP; CNP1),
Sigma; and green fluorescent protein (GFP), Invitrogen. Staining was
visualized with secondary antibodies conjugated with Alexa Fluor 488, 555,
568, 594 and 633, and confocal images were obtained using Zeiss LSM-510
confocal microscope as described previously
(Mizuguchi et al., 2001). A
cDNA probe for platelet-derived growth factor receptor 
(PDGFR) was a kind gift from W. D. Richardson (University
College London, UK).
Neurosphere culture
Neurosphere culture was established from embryonic day 13.5 (E13.5) rat
spinal cord between the upper and lower limb levels and subjected to
retrovirus infection as described previously
(Ohori et al., 2006).
Complementary DNAs (cDNAs) encoding the full-length rat Ngn1, Ngn2, Ngn3 and
mouse Pax6, Nkx2.2 and Olig2 were obtained by polymerase chain reaction,
whereas those for Mash1 (Ascl1) and Hes1, and Id1 and Id3 were provided by R.
Kageyama (Kyoto University, Japan) and T. Taga (Kumamoto University, Japan).
These cDNAs were cloned into pMXIG, and recombinant viruses
(2-4x107 colony forming unit/ml) were produced using the
packaging cell line PLAT-E. In all infection experiments, the titer of viruses
was adjusted so as to infect approximately 70% of the cells in culture. In
double-infection experiments, conditions were established for each combination
of two different viruses to ensure that more than 80% of GFP+ cells
co-expressed two transgenes simultaneously.
In the clonal assay, virus-infected cells were grown as clonal colonies in the presence of 0.8% (w/v) methylcellulose matrix for 2 weeks. The resultant colonies were collected and seeded onto glass chambers coated with poly-D-lysine (100 µg/ml) without growth factors. A week later, differentiation of neurons and glia in individual GFP+ clones was examined by in a series of triple staining for TuJ1, NG2 and GFAP. In each infection experiment, the phenotypes of at least 100 clones were examined. In the population assay, virus-infected cells were grown as a mixture for 3 days. Resultant neurospheres were dissociated into single cells and seeded onto poly-D-lysine-coated chambers. The cells were allowed to differentiate for either 4 days or 10 days.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
These molecularly distinct progenitor domains were established by E11.5 in
rats and maintained throughout the course of neuro/gliogenesis
(Fig. 1Aa-Cd,
Fig. 2Aa-Ac; see Fig. S1 in the
supplementary material). Importantly, most, if not all, VZ cells continued to
express Pax6, Olig2 or Nkx2.2 up to E16.5, just before the onset of
astrogenesis (see Fig. S2 in the supplementary material). Given their
well-established roles in pattering progenitor domains, hereafter we
collectively call these molecules `patterning factors'.
Differentiation of neurons and glia in the spinal cord begins earlier in
its rostral part, and a wave of cell genesis progresses toward caudal levels
(Novitch et al., 2003;
Diez del Corral et al., 2003).
Thus, in the same embryos, the lumbar (caudal) level represents an early
developmental stage, whereas the brachial (rostral) level represents a
slightly later stage. Comparing these two levels allowed us to correlate
precisely the temporal expression patterns of individual molecules with
specific cell genesis events.
Neurogenesis in the ventral spinal cord begins at E11.5 (roughly
corresponding to E9.5 in mice) and continues until around E16.5
(Altman and Bayer, 1984).
Proneural HLH factors of the Neurogenin (Ngn) and Mash families play crucial
roles in neurogenesis in this region
(Scardigli et al., 2001;
Parras et al., 2002). We found
that the onset and termination of Ngn expression closely paralleled the
neurogenic period in three progenitor domains. At the lumbar level, the
expression of Ngn2 in the Olig2+ domain began at E11.5 and ceased
around E14.5, which corresponded to the period of motoneuron generation
(Fig. 1Ab; see Fig. S1Ac-Cc in
the supplementary material). Likewise, Ngn1+ and Ngn2+
cells were detected in the Pax6+ domain between E11.5 and E16.5,
and Ngn3+ cells were found in the Nkx2.2+ domain between
E11.5 and E14.5 (see Fig. S1 in the supplementary material). In contrast, the
expression of Mash1 in the Olig2+ domain began at E14.5 following
the cessation of motoneuron generation
(Fig. 1Ac). Mash1+
cells emerged at E11.5 and E12.5 in the Nkx2.2+ and
Pax6+ domains, respectively, and continued until E18.5 (Figs
1,
2; see Fig. S1 in the
supplementary material). Thus, Mash1+ cells remained in the VZ
beyond the neurogenic period. Importantly, during the early neurogenic period,
the expression of these proneural HLH factors partly overlapped each other and
was, as a whole, detected only in a subset of VZ cells. Moreover, they were
always co-expressed with patterning factors (see Fig. S1 and Fig. S3 in the
supplementary material). This was in sharp contrast with the situation at the
later gliogenic period (see below).
Switch from neurogenesis to oligodendrogenesis
Early cells in the oligodendrocyte lineage retain a mitotic capacity and
thus are called oligodendrocyte precursors (OLPs)
(Noble et al., 2004). Recent
studies have shown that these OLPs migrate out from the VZ as
Nkx2.2+ or Olig2+ cells
(Soula et al., 2001;
Fu et al., 2002;
Cai et al., 2005;
Vallstedt et al., 2005;
Fogarty et al., 2005). At the
brachial level, Nkx2.2+ cells started to delaminate from the p3
domain at E12.5 (Fig. 1Aa).
About 2 days later, Olig2+ cells also began to migrate out from the
Olig2+ VZ (the p* domain at this late stage)
(Fig. 1Ba,Bb), coincided with
the onset of Olig1 expression (Fig.
1Bc). The majority of these Nkx2.2+ and
Olig2+ cells expressed the early oligodendrocyte marker O4 (see
Fig. S4 in the supplementary material). A small fraction of them, however, was
TuJ1+ neurons (see Fig. S4 in the supplementary material)
(Liu et al., 2003), indicating
that the periods of neurogenesis and oligodendrogenesis overlapped in these
ventral domains (Fig. 2E). From
E16.5 onward, Olig1+/Olig2+ and Nkx2.2+ cells
also emerged in the dorsal Pax6+ and Pax7+ domains
(Fig. 1Da-Df,Ea-Ee). These
cells also expressed O4 (Fig.
1Ef,Eg). Thus, specification of OLPs in three progenitor domains
began at different times.
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), Olig2 and
Nkx2.2 were not co-expressed at the time of specification of OLPs in rodents.
In contrast, we found that the onset of Mash1 expression closely paralleled
OLP generation in the Nkx2.2+ and Olig2+ domains
(Fig. 1Ac,Ad; see Fig. S1 in
the supplementary material). Olig1+/Olig2+ and
Nkx2.2+ cells that emerged later in the Pax6+ and
Pax7+ domains also expressed Mash1
(Fig. 1De,Df,Ec-Ee). These
migrating cells retained the expression of Mash1 in the mantle zone (MZ)
(Fig.
1Bb,Bc,Cc,Cd,De,Df,Ec-Ee). A recent fate-mapping study has shown
that these Mash1+ cells indeed differentiate into oligodendrocytes
later (Battiste et al., 2007).
Importantly, at the time when OLPs began to emerge,
Olig2+/Mash1+ and Nkx2.2+/Mash1+
cells in the Pax6+ domain did not co-express Pax6
(Fig. 1Cb,Da-Dc). This was
attributable to the downregulation of Pax6 in a subset of VZ cells, which did
not occur during earlier neurogenic periods. Thus, OLP generation in the
Pax6+ domain paralleled the loss of Pax6 expression in
Mash1+ cells (Fig.
2E).
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|
). The
expression of Id1, Id2 and Hes1, however, was already detectable at E11.5,
much earlier than the onset of astrocyte differentiation (see Fig. S5 in the
supplementary material). Their expression was separate from that of Ngns and
Mash1, but always overlapped with patterning factors during early neurogenic
periods (see Fig. S5 in the supplementary material).
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Combinatorial actions of patterning and HLH factors in vitro
The preceding study revealed dynamic changes in the co-expression patterns
of multiple transcription factors over the course of neuro/gliogenesis. We
thus speculated that their combinatorial actions could be responsible for
complex cell genesis patterns. To test this idea, we used neurosphere culture
established from E13.5 spinal cords. We used a recombinant retrovirus-mediated
overexpression system to reconstitute in vivo expression patterns of multiple
factors in neurospheres (Ohori et al.,
2006).
Multiple progenitor subtypes with distinct differentiation potentials
co-exist in the developing CNS (Noble et
al., 2004). Therefore, production of a specific cell lineage could
be regulated by multiple mechanisms. One possible scenario is that multipotent
progenitors are instructed to become lineage-restricted monopotent
progenitors, thereby producing a particular lineage. Alternatively, production
of a given lineage could be stimulated simultaneously in multiple, distinct
progenitor subtypes. To distinguish these possibilities, we performed clonal
and population assays. In the former, virus-infected cells were grown as
clonal colonies, and the differentiation repertoire of individual progenitor
clones was examined (Fig.
3A-D). Under our conditions, about 43% of control virus-infected
clones were multipotent (designated as N/O/A clones in
Fig. 3I), whereas the remaining
clones were mostly bipotent (N/O, N/A and O/A clones). In the population
assay, we compared the ratios of neurons and glia produced by a pool of
progenitors expressing different sets of transcription factors
(Fig. 3E-H). In this assay, the
percentage of TuJ1+ neurons among the total cells was highest at
day 4 after plating (DAP4) (Fig.
4), whereas differentiation of O4+ oligodendrocytes and
GFAP+ astrocytes reached plateau at DAP10 (see Fig. S6 in the
supplementary material).
Cell fate control by patterning factors
We first examined the activities of Pax6, Olig2 and Nkx2.2. None of these
factors significantly altered the percentage of multipotent clones
(Fig. 3I). Pax6, however,
increased the fraction of N/A clones at the expense of O/A clones.
Consequently, more clones contained neurons and the number of neurons per
clone increased more than tenfold compared with the control. Consistent with
this, overexpression of Pax6 in the population assay resulted in a 1.9-fold
increase in the percentage of TuJ1+ neurons
(Fig. 4D). Thus, most of the
differentiated progeny derived from Pax6-expressing progenitors were neurons
(Table 1). By contrast, Olig2
and Nkx2.2 increased the fraction of O/A clones at the expense of N/O and N/A
clones. They also increased O4+ cells and decreased
TuJ1+ cells in the population assay
(Fig. 4G,H,J,K). Consequently,
the fraction of oligodendrocytes in total differentiated cells markedly
increased (Table 1).
Importantly, in either of these cases, the overall percentage of
differentiated progeny among total cells was not markedly altered
(Table 1). Thus, patterning
factors biased the fate, but did not promote differentiation per se, of
multipotent progenitors (summarized in Fig.
8).
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Ngn2 was expressed in the Pax6+ and Olig2+ domains, whereas Ngn3 was expressed in the Nkx2.2+ domain. When tested in the clonal assay, combinatorial overexpression of these factors increased the fraction of N-only clones like Ngns alone. Cells co-expressing patterning factors, however, generated much larger colonies than those expressing Ngns alone (Fig. 3I). In line with these results, the percentages of TuJ1+ neurons were significantly smaller compared with the culture expressing Ngns alone in the population assay (Fig. 4A,D,G,J). Thus, co-expressed pattering factors attenuated the otherwise strong neurogenic and anti-proliferative activity of Ngns.
Oligodendrogenic activity of Mash1
Unlike Ngn-expressing cells, the majority (84%) of Mash1-expressing clones
became N/O-bipotent progenitors (Fig.
3I). Moreover, the numbers of neurons and oligodendrocytes
produced per clone were increased. Mash1 also increased TuJ1+ and
O4+ cells in the population assay
(Fig. 4A,B). These results are
consistent with a recent study showing that Mash1 is expressed in N/O-bipotent
progenitors in vivo (Battiste et al.,
2007).
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A striking difference was observed, however, when patterning factors were co-expressed with these inhibitory HLH factors. The majority of cells co-expressing Nkx2.2 or Olig2 remained undifferentiated (Fig. 3I, Fig. 4G-L), demonstrating that they suppressed the astrogenic activity of Id1 and Hes1. When Pax6 was co-expressed, the majority of clones became N/A progenitors. In these clones, however, the number of astrocytes produced per clone was much smaller than those in Id1- and Hes1-expressing clones and even smaller than that in the control clones (data not shown). In line with these results, over 80% of Pax6+/Id1+ and Pax6+/Hes1+ progenitors remained undifferentiated in the population assay (Fig. 4F, Table 1). In conclusion, Id1 and Hes1 promoted astrogenesis through induction of astrocyte-restricted progenitors. When co-expressed with patterning factors, however, they acted to maintain progenitors undifferentiated (Fig. 8).
Roles of Ngn2 and Mash1 in oligodendrogenesis in vivo
We next sought to ask how patterning and HLH factors control the timing of
differentiation of neurons and glia in vivo. To address this issue, we first
examined various mutant mice in which the activity and/or expression of Mash1
and Ngns were genetically modified.
Olig2+, Olig1+ and Nkx2.2+ cells migrating
in the MZ were detected at E12.5 in mice
(Fig. 5Aa-Ac). We found that
their numbers significantly decreased in Mash1-/- mice
(Fig. 5Ba-Bc,G-I).
Consequently, more Olig2+ cells remained as a tight cluster in the
VZ (compare Fig. 5Aa with Ba).
The numbers of cells expressing other OLP makers such as NG2, CNP and
PDGFR in the VZ and MZ were also decreased
(Fig. 5Ad,Ae,Bd,Be,J,K,M). Such
reduction was also detected at E14.5 and E16.5
(Fig. 5E,F; see Fig. S7 in the
supplementary material), demonstrating that the observed phenotype was not
simply a developmental delay.
|
).
Therefore, the temporal sequence of Ngn2 and Mash1 expression in the
Olig2+ domain (normally Ngn2
Mash1) was altered to
Mash1Mash1 in Ngn2KI Mash1 mice, and to
Ngn2Ngn2 + Mash1 in Mash1KI Ngn2/+ mice. We found a
larger decrease (>90%) in Olig1+ and Olig2+ cells in
Mash1KI Ngn2/+ mice than in Mash1-/-
mutants, and conversely, a 1.5-fold increase was observed in Ngn2KI
Mash1 embryos (Fig.
5Ca-Ce,Da-De,G-K). No significant increase was observed, however,
for Nkx2.2+, NG2+ or CNP+ cells in
Ngn2KI Mash1 mutants, probably because the
Nkx2.2+ domain does not normally express Ngn2.
Specification of OLPs was further examined using another set of mutant
mice. The concomitant loss of Ngn1 and Ngn2 activities results in ectopic and
precocious expression of Mash1 (Scardigli
et al., 2001). Accordingly, the number of cells expressing
PDGFR, which correspond to Olig2+ OLPs
(Fu et al., 2002), increased
about 2.5-fold in Ngn1-/-;Ngn2-/- mice
(Fig. 5L). A further increase
(4.4-fold) of PDGFR+ OLPs was detected in
Ngn1-/-;Ngn2KI Mash1 mice, in which
precocious expression of Mash1 was combined with
Ngn1-/-;Ngn2-/- mutations. These loss-
and gain-of-function studies collectively demonstrate that Mash1 plays an
important role in determining the timing of differentiation of
oligodendrocytes, and that Ngn1 and Ngn2 inhibit Mash1-dependent OLP
generation. The only partial loss of oligodendrocytes in
Mash1-/- embryos, however, suggests that molecules other
than Mash1 are also involved in oligodendrogenesis.
|
Our in vitro study suggests that Olig2+/Mash1+
progenitors generate both neurons and oligodendrocytes. Thus, we asked if a
certain neuronal subtype, in parallel to OLPs, emerges from the
Olig2+ domain in a Mash1-dependent manner. We found that a group of
Islet1+ cells, which were localized in the lateral aspect of the MZ
in the wild type, was absent in Mash1-/- embryos
(Fig. 6Aa,Ba,Ab,Bb,G). There
were two groups of cells within this cluster:
Islet1+/Olig2- and Islet1+/Olig2+
cells (Fig. 6Ab). The former
cells were dorsal interneurons derived from dorsal Mash1+
progenitors (Helms et al.,
2005; Kriks et al.,
2005), whereas the latter emerged from the Olig2+
domain. These Islet1+/Olig2+cells were negative for HB9
and O4, but were positive for TuJ1 (see Fig. S4 in the supplementary
material). They might have corresponded to a certain subtype of interneurons
or visceral motoneurons (Leber et al.,
1990; Park et al.,
2004). This Islet1+/Olig2+ cell population
was completely absent in Mash1-/- and Mash1KI
Ngn2/+ mice and, conversely, was overproduced in Ngn2KI
Mash1 mice (Fig.
6Ba-Da,Bb-Db,G).
These results collectively demonstrate that Ngn2 and Mash1 play crucial roles in the temporal control of neurogenesis and oligodendrogenesis. Precocious expression of Mash1 led to the overproduction of two late-born cell types: OLPs and Islet1+/Olig2+ neurons (Fig. 6H). Conversely, sustained expression of Ngn2 resulted in prolonged production of early-born somatic motoneurons at the expense of late-born neurons and OLPs.
Precocious oligodendrogenesis and astrogenesis in Pax6 mutants
Our in vitro data suggest that Pax6 is involved in the temporal control of
oligodendrogenesis and astrogenesis. To substantiate this idea, we examined
the onset of differentiation of these glial lineages in Pax6 mutant mice. As
shown in previous studies, the loss of Pax6 activity resulted in
downregulation of Olig2 and concomitant dorsal expansion of the
Nkx2.2+ domain (Fig.
7A,B) (Mizuguchi et al.,
2001; Novitch et al.,
2001). Consequently, more Nkx2.2+ cells and fewer
Olig1+ and Olig2+ cells were detected at E13.5 in the
ventral MZ adjacent to the p3 and p* domains of the
Pax6-/- mutant compared with the heterozygotes (+/-)
(phenotypically equivalent to the wild type)
(Fig. 7A-C,F).
We also found a small, but significant number of Olig1+, Olig2+ and Nkx2.2+ cells dorsal to the reduced p* domain in the mutant (Fig. 7A-C,G). These cells expressed the OLP marker O4 (see Fig. S4 in the supplementary material) and were detected in and adjacent to the VZ where Irx3 and Nkx6.1 were expressed, which corresponded to the normally Pax6+ domain (Fig. 7A-E, brackets). In the wild type, such cells became detectable in the Pax6+ domain only at E15.5, the time when Pax6 expression began to be downregulated. We also examined the onset of astrogenesis using glutamine synthase (GS; Glul) as an early marker for astrocytes. In the wild type, GS+ cells differentiate from E15.5 onward. In contrast, a small number of GS+ cells were already detectable at E14.5 in Pax6-/- mutants, and their number was about threefold higher than the wild type at E15.5 (Fig. 7H,I). These results demonstrate that the loss of Pax6 results in premature specification of oligodendrocytes and astrocytes (Fig. 7J).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These patterning factors participate in controlling both the timing of
differentiation and cell fate by two mechanisms. First, they act to maintain
progenitors undifferentiated by suppressing otherwise strong neurogenic and
astrogenic activities of Ngns and Id1 and/or Hes1. The suppression of the
neurogenic activity of Ngn2 by Olig2 is in accordance with the fact that the
Olig2+ domain markedly expands while producing a large number of
motoneurons (Lee et al.,
2005). Such an activity, however, is not limited to Olig2, but
common among three patterning factors. Second, three patterning factors
differentially modulate the activity of Mash1. Mash1 itself promotes
differentiation of both neurons and oligodendrocytes. Pax6, however, converts
Mash1 to become selectively neurogenic, whereas Olig2 selectively enhances
Mash1-dependent oligodendrogenesis. Thus, we propose that these two classes of
transcription factors comprise a molecular code for the coordinated
spatiotemporal control of neuro/gliogenesis. According to this model, the
relative expression levels of patterning and HLH factors at the single cell
level are crucial to determine the fate of multipotent progenitors. How the
timing and expression level of individual factors are precisely controlled
remains to be further investigated. How these two classes of transcription
factors coordinately regulate genetic programs for differentiation of neurons
and glia also needs to be examined in the future studies.
Temporal control of oligodendrogenesis
Our data suggest that different mechanisms underlie the temporal control of
oligodendrogenesis in distinct domains. Mash1 expression appears to determine
the onset of oligodendrocyte differentiation in the Nkx2.2+ and
Olig2+ domains, whereas downregulation of Pax6 in Mash1+
cells triggers their generation in the Pax6+ domain. A previous
study proposed that the co-expression of Olig2 and Nkx2.2 triggers
oligodendrogenesis in chick (Zhou et al.,
2001). In rodents, however, these proteins are not co-expressed at
the time of OLP specification (this study)
(Fu et al., 2002). Moreover,
Nkx2.2-/- mice showed a deficiency in oligodendrogenesis
only at a relatively late stage (Qi et
al., 2001). Olig1+ and Nkx2.2+ OLPs also
arose, although reduced in number, at early stages in
Olig2-/- mice (Lu et
al., 2002; Liu and Rao,
2004). Our data suggest that Nkx2.2 or Olig2 do not a priori
determine the oligodendrocyte fate, but rather control the timing of their
differentiation through the modulation of the activity of Mash1. The results
of our gain- and loss-of-function experiments in vivo and in vitro support the
idea that Mash1 acts in an instructive manner in inducting oligodendrocyte
differentiation. In Mash1-/- mice, however, OLPs were
severely reduced in number, but not completely absent. Moreover, their number
gradually recovered later in development. This could be a reason why the
deficiency in oligodendrocyte development was not recognized postnatally
(Sun et al., 1998;
Wang et al., 2001). Thus,
molecules other than Mash1 are also likely to be involved in
oligodendrogenesis.
Temporal control of astrogenesis
Previous studies suggested that astrocytes and oligodendrocytes originate
from separate progenitor domains in the spinal cord
(Lu et al., 2002;
Zhou and Anderson, 2002;
Pringle et al., 2003).
However, recent lineage-tracing studies have shown that Olig2+ and
Pax6+ domains generate both astrocytes and oligodendrocytes
(Liu and Rao, 2004;
Masahira et al., 2006;
Fogarty et al., 2005). Our data
support the idea that two glial cell lineages indeed originate from the same
progenitor domains, but the timing of their differentiation is distinct in
different domains.
Astrocytes emerge late in development. The expression of the astrogenic HLH
factors Id1 and Hes1, however, begins very early. Previous studies suggested
that proneural HLH factors account for the inhibition of astrogenesis during
early neurogenic stages (Bertrand et al.,
2002; Kageyama et al.,
2005). In fact, Ngns and Mash1 strongly suppress astrocyte
differentiation in vitro. Yet, our data have showed that the expression of
proneural and inhibitory HLH factors is mutually exclusive in the VZ,
suggesting additional mechanisms for suppression of astrogenesis during early
development. We found that these inhibitory HLH factors were always
co-expressed with patterning factors during early development, and that
astrocytes began to differentiate following the downregulation of patterning
factors in the VZ. Consistent with this, Id1 and Hes1 stimulated
differentiation of astrocytes only in the absence of Pax6, Olig2 and Nkx2.2 in
vitro. Moreover, GS+ astrocytes emerged prematurely in
Pax6-/- mice. Precocious astrogenesis has also been
reported in Olig2-/- mice
(Lu et al., 2002;
Zhou and Anderson, 2002). Yet,
accelerated differentiation of astrocytes in these mutants was rather modest.
In fact, recent studies have demonstrated that molecules other than Pax6 are
also involved in astrogenesis (Muroyama et
al., 2005; Deneen et al.,
2006). Thus, multiple mechanisms appear to underlie the temporal
control of astrocyte differentiation.
Molecular code and neural cell lineage
Several distinct models have been proposed regarding the lineage
relationship between neurons and glia. One model proposes that multipotent
progenitors are first committed to neuron- and glia-restricted progenitors,
and the latter subsequently generates astrocytes and oligodendrocytes
(Noble et al., 2004). Another
model predicts that astrocytes and oligodendrocytes develop from separate
progenitors (Lu et al., 2002;
Zhou and Anderson, 2002;
Pringle et al., 2003;
Gabay et al., 2003). The cell
genesis pattern in the ventral spinal cord, however, is not consistent with
either model. The Olig2+ domain produced neurons early, then
neurons and oligodendrocytes, and finally oligodendrocytes and astrocytes. In
contrast, in the Nkx2.2+ domain, neurons and oligodendrocytes
emerge early, and two glial subtypes differentiate later. Such domain-specific
sequences of cell genesis do not support the idea that progenitors in a given
domain are progressively committed to particular lineages. Rather, our
molecular code model suggests that cells co-expressing patterning and
inhibitory HLH factors are maintained as undifferentiated progenitors, and
that such cells sequentially generate various intermediate progenitors
destined to particular cell lineages at specific developmental timings. This
model views that at any given time point, the generation of neurons and glia
is not simply the result of a simple binary choice, like `neuron versus glia'.
A recent lineage-tracing study of Olig2+ progenitor supports a
similar model (Wu et al.,
2006). This model also suggests that undifferentiated progenitors
are preserved throughout the course of neuro/gliogenesis. In fact, a
significant number of Sox2+ undifferentiated cells remained in the
VZ at the end of neuro/gliogenesis. Such cells could persist as multipotent
cells in the postnatal and adult spinal cord. The lineage relationship between
multipotent progenitors in embryos and adults, however, remains to be further
investigated. The molecular code proposed in this study should help compare
their molecular signatures and developmental potentials.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/8/1617/DC1
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Universite Pierre et Marie Curie, IFR des Neuroscience,
Hospital de la Salpetriere, 75651 Paris Cedex 13, France
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