1 Department of Anatomical Sciences and Neurobiology, School of Medicine,
University of Louisville, Louisville, KY 40292, USA
2 Department of Medicine, University of California, San Francisco, CA 94143,
USA
3 Department of Psychiatry, University of California, San Francisco, CA 94143,
USA
4 Department of Developmental and Cell Biology, University of California at
Irvine, 4228 McGaugh Hall, Irvine CA 92697-2300, USA
Author for correspondence (e-mail:
m0qiu001{at}louisville.edu)
Accepted 10 September 2003
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SUMMARY |
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Key words: Oligodendrocyte development, Nkx6.1 mutation, In ovo electroporation, Spinal cord, Olig2, Nkx2.2
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Introduction |
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The generation of motoneurons and oligodendrocytes from the ventral spinal
cord is directly under the influence of sonic hedgehog (Shh) protein
produced in the ventral midline structures, i.e. the notochord and the floor
plate (Trousse et al., 1995;
Pringle et al., 1996
;
Poncet et al., 1996
;
Orentas et al., 1999
;
Alberta et al., 2001
;
Tekki-Kessaris et al., 2001
).
The Shh protein sets up a concentration gradient along the
ventral-to-dorsal axis of the neural tube and functions as a morphogen to
induce the expression of some members of the Nkx homeodomain
transcription factors (Qiu et al.,
1998
; Cai et al.,
1999
; Pabst et al.,
1998
; Briscoe et al.,
1999
; Briscoe et al.,
2000
) and at the same time represses the expression of other
homeodomain transcription factors (Briscoe
et al., 2000
). This combinatorial expression of the
Shh-responsive transcription factors constitutes a molecular code for
the identity of the ventral neuroepithelium
(Briscoe et al., 2000
;
Vallstedt et al., 2001
). Based
on the overlapping expression of these transcription factors, the ventral
neuroepithelium can be divided into five distinct domains (p0-p3 and pMN),
with each domain expressing a unique combination of transcription factors and
generating a particular class of neurons (V0-V3 interneurons and motoneurons)
(McMahon, 2000
;
Briscoe and Ericson, 2001
).
Motoneurons are produced from the pMN progenitor domain which expresses
Nkx6.1/Pax6/Olig2. Recent genetic studies have revealed that these
transcription factors play important roles in controlling the development of
both motoneurons and oligodendrocytes. For example, misexpression of both
Olig2 and Pax6 genes can cause aberrant development of
motoneurons and oligodendrocytes (Ericson
et al., 1997
; Sun et al.,
1998
; Sun et al.,
2001
; Zhou et al.,
2001
; Lu et al.,
2002
; Takebayashi et al.,
2002
; Zhou and Anderson,
2002
). Nkx6.1 also has an instructive role in controlling
motoneuron specification, as overexpression of the Nkx6.1 homeodomain
protein induces ectopic motoneuron formation in embryonic chicken spinal cord
(Briscoe et al., 2000
) and
mutation of Nkx6.1 leads to a drastic reduction of motoneuron genesis
(Sander et al., 2000
).
To determine the role of Nkx6.1 expression in oligodendrocyte development, we examined the specification and differentiation of oligodendrocytes in Nkx6.1 loss- and gain-of-function studies. Our studies have provided genetic and molecular evidence that Nkx6.1 regulates Olig2 expression in the ventral neuroepithelial cells and the development of oligodendrocyte progenitors. In Nkx6.1 mutant embryos, there is a delay in OPC specification and differentiation in the spinal cord, but not in the hindbrain region. Overexpression studies in chicken embryos demonstrated that Nkx6.1 can either activate or inhibit Olig2 gene expression in the developing spinal cords, depending on developmental stages.
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Materials and methods |
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In situ RNA hybridization and immunofluorescent staining
Spinal cord tissues from E10.5 to E18.5 mouse embryos were fixed directly
in 4% paraformaldehyde (PFA) at 4°C overnight. For P0 pups, animals were
fixed by cardiac perfusion with 4% PFA. Following fixation, tissues were
transferred to 20% sucrose in PBS overnight, embedded in OCT media and then
sectioned (20 µm) using a cryostat. Adjacent sections from the wild-type
and Nkx6.1 mutant embryos were subsequently subjected to in situ
hybridization or immunofluorescent staining. In situ hybridization was
performed as described in Schaeren-Wiemers and Gerfin-Moser
(Schaeren-Wiemers and Gerfin-Moser,
1993) with minor modifications, and the detailed protocol is
available upon request. Immunofluorescent procedures were previously described
in Xu et al. (Xu et al.,
2000
). Anti-Nkx2.2 (1:50, DBSH), anti-Mnr2/Hb9 (1:100; DBSH),
anti-NG2 (1:1500, Chemicon) and anti-PECAM-1 (1:50 from Pharmingen) were
obtained from commercial sources. Anti-Olig2 (1:10000) was generously provided
by Drs Chuck Stiles and David Rowitch.
Spinal cord explant culture
Segments of spinal cord tissue were isolated from E13.5 embryos at the
thoracic region and grown on 8.0 µm nucleopore polycarbonate membranes
(Costar) floating on culture medium (DMEM + N2 supplement + T3 30 ng/ml + T4
40 ng/ml + BSA 1 mg/ml + FBS 0.5% + Pen-Strep). After 7 days culture in vitro,
explants were then fixed in 4% PFA and processed for whole-mount in situ RNA
hybridization with MBP riboprobe, as described in Cai et al.
(Cai et al., 1999).
In ovo electroporation
A full-length hamster Nkx6.1 cDNA
(Rudnick et al., 1994) was
subcloned in replication-competent retroviral vector RCASBP(B)
(Morgan and Fekete, 1996
). For
in ovo electroporation, about 1.5 µl (1 µg/µl) of expression vectors
was injected into stage 11-13 (embryonic day 2 or E2) white horn chicken
embryos with the aid of Picospritzer III instrument. The injected embryos were
then subjected to three short pulses of electrical shock (25V, 50 mseconds for
each pulse) and allowed to develop for two (E4) or four (E6) more days before
they are fixed in 4% PFA for gene expression studies.
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Results |
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Our previous studies have suggested that in rodents, a majority of
Olig2+ OPCs derived from the pMN acquire Nkx2.2 expression
after they migrate into the white matter
(Fu et al., 2002).
Interestingly, upregulation of Nkx2.2 expression in the white matter
OPCs occurred only in the wild-type but not mutant spinal cords
(Fig. 3E,F). Double
immunolabeling confirmed that all Nkx2.2+ cells in the white matter
co-expressed Olig2 (Fig.
3I,J). Together, these results indicated a delayed or defective
Nkx2.2 upregulation in Olig2+ OPCs in Nkx6.1 mutant
spinal cords.
Myelin gene expression is delayed and reduced in Nkx6.1 mutants
Recent studies have demonstrated that the Nkx2.2 expression in
white matter precedes and regulates oligodendrocyte differentiation
(Qi et al., 2001;
Sun et al., 2001
;
Zhou et al., 2001
;
Fu et al., 2002
). Thus, the
absent Nkx2.2 expression in the white matter of mutant spinal cords
would predict retarded oligodendrocyte differentiation and maturation. To
examine this possibility, we investigated the expression of the mature
oligodendrocyte markers myelin basic protein (MBP) and proteolipid protein
(PLP) in mutant spinal cords at the thoracic level. At E17.5, a small number
of MBP+ oligodendrocytes were found in the ventral spinal cord of wild-type
embryos, but not in the mutants (Fig.
4A,B). By P0, although the number of MBP+ oligodendrocytes in
wild-type mice increased, MBP+ and PLP+ oligodendrocytes were still not
detected in the mutant spinal cords (Fig.
4C-F). Unfortunately, further development of oligodendrocytes in
postnatal mutant cords can not be assessed owing to the neonatal death of
mutant animals.
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Regulation of Olig2 and Sox10 gene expression by Nkx6.1 in embryonic
chicken spinal cord
Recent studies have indicated that Olig1 and Olig2 genes
are required for the initial specification of the oligodendrocyte lineage in
the pMN domain (Lu et al.,
2002; Zhou et al., 2002), raising the possibility that the effects
of the Nkx6.1 mutation on oligodendrocyte development in the spinal
cord is mediated by its direct regulation of Olig gene expression in the
ventral neuroepithelium. To investigate whether expression of Nkx6.1
can activate Olig gene expression and oligodendrogenesis in the developing
spinal cord, we overexpressed Nkx6.1 gene in stage 11-13 (E2) chicken
spinal cord by in ovo electroporation and then examined its effects on the
expression of Olig2 and oligodendrocyte marker gene Sox10.
As a positive control, the motoneuron marker Mnr2/Hb9 (Hlxb9
Mouse Genome Informatics) was also included in the assay
(Tanabe et al., 1998
). Two
days after Nkx6.1 electroporation (at E4), ectopic expression of
Olig2, Mnr2/Hb9 and Sox10 was all induced in the
electroporated side of the spinal cords
(Fig. 6A-D; see Fig. S2 at
http://dev.biologists.org/supplemental/).
Normally, expression of Sox10 in the ventral neuroepithelium is not
detectable until E6 (Fig. 6H).
Thus, overexpression of Nkx6.1 can induce precocious expression of
oligodendrocyte marker Sox10. However, induction of other markers
such as O4 and MBP was not observed at this stage (data not shown), possibly
owing to the lack of expression of the co-factor Nkx2.2
(Zhou et al., 2001
).
Surprisingly, 4 days after electroporation, no ectopic expression of
Olig2 and Sox10 was observed in the electroporated side of
the spinal cords, although ectopic expression of Mnr2/Hb9 remained in
the adjacent section (Fig.
6E-H). Moreover, the endogenous expression of Olig2 in
the ventral neuroepithelium was significantly reduced by Nkx6.1
overexpression (Fig. 6F; see
Fig. S2 at
http://dev.biologists.org/supplemental/).
These results suggest that Nkx6.1 can function as either an activator
or an inhibitor of Olig2 gene expression, depending on the
developmental stage of the spinal cord tissue.
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Discussion |
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Consistent with our hypothesis that Nkx6.1 regulates spinal
oligodendrogenesis by controlling Olig expression, overexpression of
Nkx6.1 in embryonic chicken spinal cords lead to ectopic expression
of Olig2, Mnr2 and the OPC marker Sox10 at E4, 2 days after
in ovo electroporation (Fig. 6,
Fig. S1 at
http://dev.biologists.org/supplemental/).
Ectopic Sox10 expression occurred 2 days earlier than its endogenous
expression, which is not detectable until E6. This precocious induction of an
OPC lineage marker probably resulted from the high level of Nkx6.1
protein expression or activity in our electroporation studies. However,
ectopic expression of later oligodendrocyte markers was not induced at this
stage, possibly because it requires the co-expression of Nkx2.2
(Zhou et al., 2001) or a
longer induction of Olig2 by Nkx6.1. Unexpectedly, 4 days
after electroporation, the ectopic expression of Olig2 and
Sox10 was not detected and the endogenous expression of
Olig2 in the pMN domain was markedly inhibited
(Fig. 6, see Fig. S2 at
http://dev.biologists.org/supplemental/).
The rapid downregulation of Olig2 in Nkx6.1-overexpressing
cells after E4 might explain why we did not observe a further increase of
oligodendrogenesis at E6 and later stages in the
Nkx6.1-electroporated embryos. Thus, Nkx6.1 appears to
switch its role from an activator to an inhibitor of Olig2 expression
at later stages, possibly owing to the dynamic expression of its co-factors.
This role switch might also explain why Nkx6.1 and Olig2 are
not expressed in the same cells after oligodendrogenesis. Nkx6.1
expression is immediately downregulated in the Olig2+ OPC cells after
they migrate out of the ventricular zone
(Xu et al., 2000
). By
contrast, Nkx6.1 expression is retained in the ventricular cells
throughout embryonic and postnatal animal development
(Fu et al., 2003
), while
Olig2 expression is rapidly lost in the ventricular neuroepithelial
cells after oligodendrogenesis stages (E9 in chicken or E16.0 in mouse).
Interestingly, the ventricular Olig2 expression in Nkx6.1
mutant spinal cords is not increased or prolonged after oligodendrogenesis.
One possible explanation is that the Olig2+ ventricular cells are
prematurely depleted during oligodendrogenesis in the mutants because of its
small pool size (Fig. 1B,D,F),
before Nkx6.1 becomes inhibitory. In addition, the rapid and
premature downregulation of Olig2 expression at E6 in our
Nkx6.1 overexpression studies is likely to be due to an unregulated
high-level Nkx6.1 protein expression or activity.
The stage-dependent regulation of gene expression was previously suggested
for other homeodomain transcription factors such as Nkx2.2. During
early neurogenesis stages, Nkx2.2 inhibits Olig2 expression
in the pMN domain; however, during oligodendrogenesis stages, Nkx2.2
and Olig2 are co-expressed in the same OPC cells and Nkx2.2
becomes a co-activator, instead of an inhibitor, of Olig2
(Qi et al., 2001;
Zhou et al., 2001
).
Identity of Nkx2.2-expressing cells in embryonic mouse spinal
cord
Recent molecular and genetic studies have established that the
Olig2+/Pdgfra+ OPC cells originate from the pMN domain
(Sun et al., 1998;
Richardson et al., 2000
;
Fu et al., 2002
;
Lu et al., 2002
;
Zhou and Anderson, 2002
). In
rodents, the majority of Olig2+ OPCs appear to acquire
Nkx2.2 expression after they migrate out into the white matter prior
to their terminal differentiation (Fu et
al., 2002
). This study has provided additional support for this
concept, as the delayed generation of Olig+/Pdgfra+ OPCs in
Nkx6.1 mutant spinal cords is associated with a delay of
Nkx2.2 expression in the white matter
(Fig. 3).
It has also been proposed that a second source of OPCs might be generated
from the Nkx2.2+ p3 domain of ventral neuroepithelium as well
(Soula et al., 2001;
Zhou et al., 2001
;
Fu et al., 2002
;
Lee et al., 2003
). Based on
the migration pattern of Nkx2.2+ cells, it was hypothesized that OPC
cells derived from the p3 domain are initially
Nkx2.2+/Pdgfra-/Olig2-, but become Olig2+ after they migrate
into the white matter (Fu et al.,
2002
). However, in contrast to the previous hypothesis, this group
of Nkx2.2 cells did not appear to disperse into the white matter, and
nor did they gain Olig2 expression
(Fig. 3I,J). Instead, they
remained in the ventral gray matter and started to co-express the panneuronal
marker NeuN at later stages of embryogenesis
(Fig. 3K,L; see Fig. S1 at
http://dev.biologists.org/supplemental/).
Collectively, these observations would strongly argue that Nkx2.2+
cells derived from the p3 domain develop into neurons (possibly V3 ventral
interneurons), rather than OPC cells. This new interpretation is consistent
with the recent genetic evidence that oligodendrocyte development in embryonic
mouse spinal cord is coupled to motoneuron development
(Lu et al., 2002
;
Zhou and Anderson, 2002
).
Region-specific regulation of Olig2 expression and oligodendrogenesis
by Nkx6.1 expression
Previous expression studies have demonstrated that Nkx6.1 is also
expressed in the ventral hindbrain, including motoneuron progenitor cells
(Qiu et al., 1998;
Puelles et al., 2001
;
Takahashi and Osumi, 2002
).
Loss of Nkx6.1 gene activity disrupts the development of somatic
motoneurons derived from the Olig1/2+ neuroepithelium
(Sander et al., 2000
).
Surprisingly, unlike in the spinal cord, the ventral expression of Olig genes
in the mutant hindbrain is not altered during oligodendrogenesis stages. The
generation of Pdgfra+ and Olig1/2+ OPCs in this region
appears to be normal and their differentiation and maturation are also on
schedule in Nkx6.1 mutants. One possible explanation for the
differential effects of Nkx6.1 mutation on oligodendrogenesis in the
ventral spinal cord and hindbrain is that the loss of Nkx6.1 is
compensated by Nkx6.2, given that Nkx6.1 and Nkx6.2
have a similar expression pattern in the hindbrain but not in the spinal cord
(Cai et al., 1999
;
Vallstedt et al., 2001
).
However, our preliminary studies have indicated that oligodendrocyte
development in the hindbrain is not significantly compromised in the
Nkx6.1/Nkx6.2 double mutants (data not shown). Together, our studies
indicate a regional difference in the regulation of Olig2 expression
and oligodendrocyte development by the Nkx6.1 homeodomain
transcription factor.
Consistent with the previous suggestions on multiple origins of OPCs in the
developing hindbrain (Spassky et al.,
1998; Spassky et al.,
2000
; Davies and Miller,
2001
), Olig1 and Olig2 are expressed in multiple
ventricular and subventricular zones in the rostral hindbrain (metencephalon)
at E13.5 (Fig. 8A-D), and OPC
cells can be produced from the neuroepithelial cells both inside and outside
the Nkx6.1-expressing domain (Figs
8,
9). The lack of Nkx6.1
expression in the dorsal metencephalon, together with the normal production of
OPC cells from Nkx6.1 mutant hindbrain tissue, indicates the
existence of an Nkx6.1-independent mechanism for regulation of Olig
gene expression and oligodendrogenesis in the rostral CNS. This is
particularly true for the developing telencephalon, where Nkx6.1 is
not expressed and oligodendrogenesis occurs normally in Nkx6.1 mutant
embryos (data not shown). In addition, the generation of OPC cells, but not
motoneurons, from the dorsal Nkx6.1-negative neuroepithelial cells in
the rostral hindbrain would also indicate the lack of an obligatory lineage
relationship between oligodendrocytes and motoneurons in this region.
Regulation of oligodendrocyte differentiation and maturation
Previous studies have demonstrated that the Olig2+/Pdgfra+ OPC
cells originate from the pMN domain (Sun
et al., 1998; Richardson et
al., 2000
; Fu et al.,
2002
), and acquire Nkx2.2 expression as they migrate into
the white matter prior to their terminal differentiation
(Fu et al., 2002
). The
up-regulation of Nkx2.2 expression in OPC cells is required for
normal myelin gene expression and oligodendrocyte differentiation
(Qi et al., 2001
;
Sun et al., 2001
;
Zhou et al., 2001
). In
Nkx6.1 mutant spinal cords, the delayed production of OPC cells is
accompanied by a delayed expression of Nkx2.2 in the white matter
(Fig. 3) and myelin genes
MBP/PLP (Figs 4,
5).
The mechanisms underlying the parallel defect of oligodendrocyte
specification and differentiation remain to be determined at this stage. As
Nkx6.1 per se is not expressed in migratory OPC cells
(Xu et al., 2000) and the OPCs
from the Nkx6.1 mutant spinal cord are capable of differentiating
into MBP+ mature oligodendrocytes both in vitro
(Fig. 4G,H) and in vivo
(Fig. 5), the effect of
Nkx6.1 mutation on oligodendrocyte differentiation is likely to be
indirect or cell non-autonomous. One possibility is that oligodendrocyte
differentiation in the spinal cord is controlled by an intrinsic clocking
mechanism, as suggested by previous studies on the in vitro differentiation of
OPC cells (Temple and Raff,
1986
; Gao et al.,
1997
). It is possible that during development, OPC cells also exit
the cell cycle and undergo terminal differentiation after a fixed number of
cell divisions or a fixed amount of time after birth, and a delay of
progenitor production is coupled with a delay of differentiation. Another
possibility is that the decreased expression of neuregulin 1 (Nrg1) is
responsible for the retarded oligodendrocyte differentiation in the spinal
cord (Vartanian et al., 1999
;
Park et al., 2001
). In
Nkx6.1 mutants, motoneuron development is inhibited
(Sander et al., 2000
) and the
production of Nrg1 by motoneurons is markedly reduced (data not shown).
Finally, the terminal differentiation of oligodendrocytes could be regulated
by a density-dependent mechanism. It is conceivable that OPC cells may secrete
some autocrine differentiation-inducing factor whose concentration could be
reduced in the mutants as compared with wild-type embryos, because there are
fewer cells in the prenatal period. Future studies are needed to unravel the
molecular mechanisms underlying the delayed oligodendrocyte differentiation
and maturation in Nkx6.1 mutant spinal cords.
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ACKNOWLEDGMENTS |
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Footnotes |
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* These authors contributed equally to this work
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