Differential modulation of BMP signaling promotes the elaboration of cerebral cortical GABAergic neurons or oligodendrocytes from a common sonic hedgehog-responsive ventral forebrain progenitor species.

Abstract

During cerebral cortical development, excitatory glutamatergic projection neurons are generated from neural stem cells intrinsic to the early embryonic cortical ventricular zone by a process of radial migration, whereas most inhibitory gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes (OLs) appear to be elaborated from ventral forebrain stem cells that initially undergo tangential cortical migration before terminal lineage maturation. In contrast to the more compartmentalized developmental organization of the spinal cord, the generation of neurons and OLs from a common ventral forebrain stem cell would expose these cells to the sequential actions of ventral and dorsal gradient morphogens [sonic hedgehog (Shh) and bone morphogenetic proteins (BMPs)] that normally mediate opposing developmental programs. Here we report that Shh promotes GABAergic neuronalOL lineage restriction of forebrain stem cells, in part, by activation of the basic helix-loop-helix transcription factors, Olig2 and Mash1. In mutant mice with a generalized defect in tangential cortical migration (Dlx12--), there is a profound and selective reduction in the elaboration of both cortical GABAergic neurons and OLs. Our studies further demonstrate that the sequential elaboration of cortical GABAergic neurons and OLs from common Shh-responsive ventral forebrain progenitors requires the spatial and temporal modulation of cortical BMP signaling by BMP ligands and the BMP antagonist, noggin, respectively. These findings suggest an integrative model for cerebral cortical GABAergic neuronal and OL lineage maturation that would incorporate the sequential contributions of the ventral and dorsal forebrain, and the potential role of regional developmental cues in modulating transcriptional codes within evolving neural lineage species.

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Differential modulation of BMP signaling promotes
the elaboration of cerebral cortical GABAergic
neurons or oligodendrocytes from a common
sonic hedgehog-responsive ventral forebrain
progenitor species
Shau-Yu Yung*†, Solen Gokhan†‡§, Jennifer Jurcsak§, Aldrin E. Molero*, Joseph J. Abrajano*, and Mark F. Mehler*‡§¶?**
‡F. M. Kirby Program in Neural Protection and Repair, Departments of§Neurology, *Neuroscience, and¶Psychiatry and Behavioral Sciences, Rose F. Kennedy
Center for Research in Mental Retardation and Developmental Disabilities,?Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine,
Bronx, NY 10461
Communicated by Dominick P. Purpura, Albert Einstein College of Medicine, Bronx, NY, September 30, 2002 (received for review July 8, 2002)
During cerebral cortical development, excitatory glutamatergic
projection neurons are generated from neural stem cells intrinsic
to the early embryonic cortical ventricular zone by a process
of radial migration, whereas most inhibitory ?-aminobutyric acid
(GABA)ergicinterneuronsandoligodendrocytes(OLs)appeartobe
elaborated from ventral forebrain stem cells that initially undergo
tangential cortical migration before terminal lineage maturation.
In contrast to the more compartmentalized developmental orga-
nizationofthespinalcord,thegenerationofneuronsandOLsfrom
a common ventral forebrain stem cell would expose these cells to
the sequential actions of ventral and dorsal gradient morphogens
[sonic hedgehog (Shh) and bone morphogenetic proteins (BMPs)]
that normally mediate opposing developmental programs. Here
we report that Shh promotes GABAergic neuronal?OL lineage
restriction of forebrain stem cells, in part, by activation of the basic
helix–loop–helix transcription factors, Olig2 and Mash1. In mutant
mice with a generalized defect in tangential cortical migration
(Dlx1?2???), there is a profound and selective reduction in the
elaboration of both cortical GABAergic neurons and OLs. Our
studies further demonstrate that the sequential elaboration of
cortical GABAergic neurons and OLs from common Shh-responsive
ventral forebrain progenitors requires the spatial and temporal
modulation of cortical BMP signaling by BMP ligands and the BMP
antagonist, noggin, respectively. These findings suggest an inte-
grative model for cerebral cortical GABAergic neuronal and OL
lineage maturation that would incorporate the sequential contri-
butions of the ventral and dorsal forebrain, and the potential role
of regional developmental cues in modulating transcriptional
codes within evolving neural lineage species.
T
glial lineage diversity within the evolving mammalian cerebral
cortex (1–4). During early embryonic development, excitatory
glutamatergic projection neurons are elaborated from neural
stem cells after radial migration from the cortical ventricular
zone (VZ). By contrast, more recent slice culture cell migration
studies and additional analyses using mutant mice with defects
in tangential cortical migration (Dlx1?2???) and in regional
progenitor domain specification (Nkx2.1???) have demon-
strated that most cortical inhibitory ?-aminobutyric acid
(GABA)ergic interneurons are generated from stem cells lo-
cated within the medial ganglionic eminence (MGE) and lateral
ganglionic eminence (LGE) of the ventral forebrain after sep-
arate waves of tangential cortical migration (5). Although the
majority of these migratory cells undergo rapid cortical disper-
sion and the elaboration of different GABAergic neuronal
subtypes, a smaller proportion of these cells are retained within
here is an emerging consensus that two distinct developmen-
tal pathways are required for the generation of neuronal and
the late embryonic cortical subventricular zone (SVZ), where
they sequentially give rise to GABAergic neurons and additional
cells that become mitotically active during the phase of perinatal
cortical gliogenesis (5). In fact, clonal analysis studies have
suggested that the ventral forebrain may contain common
GABAergic neuronal?oligodendrocyte (OL) progenitor cells
(6), and in this scenario the mitotically active SVZ cells may
therefore represent proliferating OL precursors. However, in
these studies, the putative contribution of ventral forebrain
GABAergic neuronal?OL progenitors to cerebral cortical neu-
rogenesis and gliogenesis was inferred from the presence of
Dlx-immunoreactivity in a subset of cortical OL species (6)
rather than confirmed by the use of additional genetic or
developmentalanalyses.Inthecurrentstudies,wehavefirstused
comparative clonal analysis of premigratory stage ventral and
dorsal forebrain and postmigratory stage cerebral cortical pro-
genitors and complementary genetic analysis using Dlx1?2 mu-
tant mice to demonstrate that the ventral forebrain does, in fact,
contain common GABAergic neuronal?OL progenitors capable
of giving rise to cerebral cortical GABAergic neurons and OLs.
The molecular mechanisms that orchestrate cerebral cortical
neurogenesis and gliogenesis, particularly with regard to the
distinctive contribution of the ventral forebrain tangential mi-
gratory pathway are poorly understood (2–4, 7, 8). Throughout
the neuraxis, sonic hedgehog (Shh) and bone morphogenetic
proteins (BMPs) are ventral and dorsal gradient morphogens,
respectively (8–11). These soluble signaling molecules specify
discrete regional progenitor domains by the concentration-
dependent elaboration of contiguous pairs of homeodomain and
basic helix–loop–helix (bHLH) transcription factors that exhibit
mutually antagonistic interactions necessary to refine individual
progenitor domains and to promote the timely elaboration of
specific neuronal and glial subtypes (12). In the spinal cord,
astrocytes are generated from dorsal domains where BMP
signaling predominates, whereas OLs are generated from a
circumscribed ventral progenitor domain that initially gives rise
to motor neurons under the influence of specific levels of Shh
signaling (10, 12). Within the motor neuron domain, Olig2, the
only bHLH member of this elaborate progenitor code, is essen-
tial for linking a specific neuronal subtype to an OL specification
program and for coordinating the acquisition of pan-neuronal
Abbreviations:BMP,bonemorphogeneticprotein;OL,oligodendrocyte;Shh,sonichedge-
hog; N-Shh, N-terminal Shh; VZ, ventricular zone; MGE, medial ganglionic eminence; LGE,
lateral ganglionic eminence; SVZ, subventricular zone; bHLH, basic helix–loop–helix; EGF,
epidermal growth factor; FGF, fibroblast growth factor.
†S.-Y.Y. and S.G. contributed equally to this work.
**To whom correspondence should be addressed. E-mail: mehler@aecom.yu.edu.
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traits and motor neuron subtype specification by promoting the
expression of Neurogenin 2 (13–16). Neurogenin 2, in turn,
limits the number of progenitor cell cycle divisions and initiates
a downstream transcriptional cascade that reinforces the neu-
ronal subtype specific developmental program. The later elab-
oration of OL species from this progenitor domain is mediated,
in part, by the dorsal shift of Nkx2.2 into the Olig2??Nkx2.2?
domain through a mechanism that may involve transient Shh
signaling from the floor plate (13–16). However, genetic and
developmental analyses suggest that Olig1 rather than Nkx2.2 is
required for OL specification, whereas Nkx2.2 is necessary for
OL terminal maturation (17, 18). In addition, the neuronal?glial
switch requires the extinction of Neurogenin2 before the ex-
pression of Olig1 within the Olig2? progenitor domain (13–16).
The role of soluble signaling molecules and regional progenitor
transcriptional codes in orchestrating related developmental
processes during forebrain lineage specification and maturation
are less well defined.
The developmental organization of the forebrain differs from
the spinal cord in several important respects. First, GABAergic
neurons rather than motor neurons are coupled to OLs (6, 8). In
addition, GABAergic neurons are known to express the bHLH
transcription factor, Mash1 rather than Neurogenin2, and
Mash1 persists in OL species after lineage specification (8,
19–22). Further, because Olig2 is known to couple specific
neuronal subtypes to OLs (23, 24), and Olig2 and Mash1 are
preferentially expressed within the ventral forebrain before
tangential cortical migration (21, 22, 25), it is likely that Olig2
and Mash1 are expressed in a common GABAergic?OL pro-
genitor before tangential cortical migration. Finally, initial ven-
tral forebrain specification and tangential cortical migration
would expose these bipotent progenitors to sequential ventral
anddorsalgradientmorphogensthatnormallymediateopposing
developmental programs (8, 26). These cumulative observations
suggest that Shh and BMPs may exhibit complementary and
cooperative roles in cerebral cortical GABAergic neuronal and
OL lineage elaboration. During nervous system development,
regional neural stem cells give rise to differentiated progeny
through a process of progressive lineage restriction and the
formation of intermediate progenitor species that have different
responseprofilestoenvironmentalcues(27).Ourcurrentstudies
indicate that Shh is required for the generation of GABAergic
neuronal?OL progenitors from ventral forebrain stem cells
through the mediation of Olig2 and Mash1.
Prior studies from our laboratory and those of others have
shown that BMP ligands are expressed within the early embry-
onic cerebral cortex at the time of GABergic neuronal lineage
specification, whereas the BMP inhibitor, noggin, is selectively
expressed within the late embryonic and perinatal subcortical
white matter, the site of subsequent OL lineage specification and
maturation(28–30).Additionalstudiesfromourlaboratoryhave
shown that BMP ligands inhibit the initial lineage specification
but subsequently promote the migration and differentiation of
early-born (glutamatergic) neurons from cortical VZ progenitor
cells (28, 29). Further, BMP2 has been shown to promote the
elaborationofstriatalGABAergicneurons(31).Finally,wehave
demonstrated that OL lineage elaboration requires active inhi-
bition of BMP signaling (28, 32). These cumulative observations
suggest that spatiotemporal modulation of BMP signaling may
be important for the sequential elaboration of cortical GABAer-
gic neurons and OLs from lineage-restricted ventral forebrain
progenitors initially specified by regional Shh signaling. Our
current studies demonstrate that BMP2 and noggin potentiate
the elaboration of GABAergic neurons and OLs, respectively,
from ventral forebrain progenitor cells previously exposed to
specific levels of endogenous Shh signaling, and also from
premigratory stage dorsal forebrain cells that have undergone a
dorsal-to-ventral fate switch in response to exogenous Shh
signaling.BecausedifferentialmodulationofBMPsignalingmay
alter the profiles of expression of transcription factors involved
in neural lineage decisions (7, 8), the precise developmental
regulation of cortical BMP signaling molecules may be essential
for orchestrating GABAergic neuronal and OL lineage elabo-
ration by promoting changing profiles of transcriptional codes
within sequential stage-specific progenitor species.
Materials and Methods
Neural Cell Cultures. Dlx1?2 mutant mice were provided by
J. L. R. Rubenstein (University of California, San Francisco).
Timed pregnant CD1 and Dlx1?2 mutant mice were killed at
embryonic days (E) 12.5 and 16.5. Tissue samples from the
embryonic murine cortex, the MGE, and the LGE were carefully
separated and mechanically dissociated. Primary cultures were
generated by plating dissociated cells at clonal density (15–40
cells?cm2) in culture wells containing poly-D-lysine (PDL, 20
?g?ml)-coated glass coverslips and propagated in serum-free
media (SFM) consisting of DMEM?F12 (Life Technologies,
Grand Island, NY), L-glutamine (2 mM), penicillin (100 units?
ml), N2 and B27 (Life Technologies) supplements, and basic
fibroblast growth factor (bFGF, 1 ng?ml; Collaborative Biomed-
ical Products, Bedford, MA). Primary cultures were normally
propagated for 6 days in vitro (DIV), and subsequently fixed and
processed for immunocytochemical analysis. For specific exper-
imental conditions, the active N-terminal form of Shh (N-Shh,
1–250 ng?ml; Curis, Cambridge, MA), BMP2 (10 ng?ml;
Genetics Institute, Cambridge, MA) or noggin (10 ng?ml; Re-
generon Pharmaceuticals, Tarrytown, NY) were added at the
time of initial plating. Self-renewing, multipotent stem cells were
generated from E12.5 cerebral cortical cells by clonal expansion
(5–15 clones per 35 cm2) in the absence of polycationic substrate
in SFM containing bFGF (10 ng?ml) and heparin (2 ?g?ml) and
propagated for 3 DIV. For examination of the combinatorial
effects of soluble factors (N-Shh, 50 ng?ml) and specific gene
manipulation paradigms (Olig2 and Mash1, see below) on clonal
expansion and on the profiles of lineage elaboration, Shh and?or
the appropriate adenoviral vectors or antisense oligonucleotides
were added concurrently at the time of initial FGF application.
For examination of progenitor cell lineage potential, individual
intact cellular clones were transferred to separate culture wells
on PDL-coated glass coverslips and allowed to differentiate in
SFM containing bFGF (1 ng?ml) for an additional 6 DIV.
Adenovirus Construction. An adenoviral vector expressing both
Olig2 and GFP under separate cytomegalovirus (CMV) pro-
moterswasconstructedwiththeAdEasysystem(33).Full-length
Olig2 cDNA was generated by RT-PCR using the murine Olig2
cDNA sequence from GenBank (accession no. AB038697). All
plasmids for the adenoviral construction were provided by
B. Vogelstein (Johns Hopkins University, Baltimore). At the
time of plating, progenitor culture were infected with control
GFP-expressing and with Olig2?GFP-expressing adenoviruses.
Optimal conditions were defined by a multiplicity of infection
(MOI) of 50.
Antisense Oligonucleotide Inhibition of Gene Expression in Neural
Progenitor Cells. Complete inhibition of protein expression by
phosphorothioate-modified antisense oligonucleotides was ver-
ified by using Western analysis. The following oligonucleotides
(finalconcentration,5?M)wereaddedatthetimeofplatingand
reintroduced when detectable levels of protein expression re-
emerged: Olig2 antisense: 5?-CGTCCGAGTCCATGGCCA-3?;
Olig2 sense: 5?-TGGGCCATGGACTCGGACG-3?; Mash1
antisense; 5?-CCGGGCCGTACCTCTC-3?; Mash1 sense:
5?-GGCCCGGCATGGAGAG-3?.
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Immunocytochemistry. Cellular preparations were fixed and pro-
cessed for immunofluorescence microscopy as described (32).
Primary antibodies were diluted to optimal concentrations in
PBS: nestin (Sigma), 1:1,000; ?-tubulin (Sigma), 1:400; gluta-
mate (Sigma), 1:2,000; GABA (Sigma), 1:2,500; GAD65
(Chemicon) 1:1,000; O4 supernatant from mouse hybridoma cell
line (S. Pfeiffer, University of Connecticut), 1:5; Olig1 (R. Lu,
D. Rowitch, and C. Stiles, Dana–Farber Cancer Institute, Bos-
ton) 1:100; Olig2 (H. Takebayashi, Okazaki National Institute,
Okazaki, Japan, and M. Nakafuku, University of Tokyo, Tokyo),
1:200; glial fibrillary acidic protein (GFAP) (Sigma), 1:400;
Mash1 (PharMingen), 1:250; Ngn1 (Chemicon), 1:100; Nkx2.2
(DevelopmentalStudiesHybridomaBank,IowaCity,IA),1:250.
Statistical Analysis. Clonal lineage composition was estimated by
counting a minimum of 32 clones from five defined fields (cm2)
from 24-well tissue culture plates and was verified a minimum of
fourtimes.Experimentalvaluesrepresenttheproportionoftotal
clones containing the appropriate lineage markers as a percent-
age with a 95% confidence interval. The absolute proportion of
target clones between control and experimental conditions were
compared by using ?2analysis. Significant differences were
considered at a P value ?0.05.
Results and Discussion
Developmental and Genetic Evidence That Ventral Forebrain Progen-
itor Cells Give Rise to Cerebral Cortical GABAergic Neurons and OLs.
We first examined the ability of cultured progenitor cells derived
fromtheearlyembryonicventralanddorsalforebraintogiverise
to GABAergic neurons and OL species. Before tangential
cortical migration, individual MGE and LGE progenitors of the
ventral forebrain but not age-matched cerebral cortical progen-
itors have the potential to generate clones containing both
GABAergic neurons and OL species as well as pure GABAergic
neuronal and OL clones (Fig. 1A). By contrast, after tangential
migration from the MGE and the LGE, individual cerebral
cortical progenitors develop the propensity to generate clones
containing both GABAergic neurons and OL progenitors and
also pure GABAergic neuronal and OL clones (Fig. 1A) con-
taining cells derived from the ventral forebrain (Dlx?, not
shown) (34). These observations suggest that after tangential
cortical migration, individual progenitor cells derived from the
ventral forebrain have the potential to give rise to both cortical
GABAergic neurons as well as OLs. To further confirm the role
of tangential cortical migration in the elaboration of cortical
GABAergic neurons and OLs, we performed similar experi-
mental paradigms using mutant mice that display a generalized
defect in tangential cortical migration (Dlx1?2???). Previous
studies have shown that Dlx1?2??? mice have profound reduc-
tions in cortical GABAergic neurons, but the fate of OL species
in cortical cells derived from these mutant mice have never been
previously examined (5). Individual Dlx1?2??? progenitors
from the premigratory stage ventral forebrain displayed a sig-
nificant increase in the elaboration of clones containing both
GABAergicneuronsandOLspecies(Fig.1B).Bycontrast,there
was a profound reduction in the elaboration of clones containing
both GABAergic neurons and OLs and a corresponding signif-
icant increase in the generation of clones containing glutama-
tergic neurons from individual Dlx1?2??? postmigratory stage
cerebral cortical progenitors (Fig. 1C). Analogous postmigra-
tory stage cortical cells from Dlx1?2??? progenitors exhibited
more limited reductions in the generation of GABAergic neu-
rons and OLs (Fig. 1C), indicating Dlx gene dosage effects.
Comparative analysis of perinatal (P0) tissue sections from
Dlx1?2??? and Dlx1?2??? mice confirmed the developmen-
tal stage-specific reduction in cortical GABAergic neurons and
OL precursors (data not shown). These cumulative observations
suggest that disruption in the process of tangential cortical
migration is responsible for the profound impairment in the
generation of cortical GABAergic neurons and OLs, and may
result in compensatory increases in the proportion of glutama-
tergic neurons. These findings provide additional genetic evi-
dence that cortical GABAergic neurons and OLs are derived
from the ventral forebrain, and further suggest that a combina-
torial transcriptional code is responsible for the generation of
these specialized cortical neuronal and glial subtypes (8, 35).
Shh Promotes GABAergic Neuronal?OL Lineage Restriction of Early
Embryonic Forebrain Stem Cells and the Selective Induction of Tran-
scription Factors Involved in GABAergic Neuronal and Oligodendro-
glial Lineage Specification and Maturation. Previous studies have
shown that the early embryonic cerebral cortex contains both
epidermal growth factor (EGF)- and FGF-responsive stem cells
(36). Because Shh is already expressed by the early gastrula stage
and ventral forebrain Shh-responsive progenitors are initially
derived from FGF-responsive stem cells (4, 8), we elected to
examine the effects on neural lineage potential of naive dorsal
forebrain FGF-responsive stem cells after exposure to Shh.
When individual primary early embryonic cerebral cortical cells
underwent clonal expansion in the presence of bFGF and
Fig. 1.
to both GABAergic neurons and OLs that populate the postmigratory stage cerebral cortex. (A) The proportion of total clones containing GABAergic neurons,
OL species, or GABAergic neurons and OL species generated after propagation of primary E12.5 MGE and LGE and E12.5 and E16.5 cerebral cortical (Ctx)
progenitors for 6 DIV. All data points represent the proportion of target clones with its 95% confidence interval. (B) The proportion of clones containing
GABAergic neurons and OL species generated after propagation of primary E12.5 Dlx1?2???, Dlx1?2???, and Dlx1?2??? MGE and LGE progenitors for 6 DIV.
Statistical analysis was calculated by using ?2analysis:*, P ? 0.001 (compared with the E12.5 Dlx1?2??? MGE clonal condition); ?, P ? 0.001 (compared with
the E12.5 Dlx1?2??? LGE clonal condition). (C) The proportion of total clones containing both GABAergic neurons and OL species or glutamatergic neurons
generatedafterpropagationofprimaryE16.5Dlx1?2???,Dlx1?2???,andDlx1?2???cerebralcorticalprogenitorsfor6DIV.Statisticalanalysiswascalculated
by using ?2analysis:*, P ? 0.001 (compared with the Dlx1?2??? GABA?O4 clonal condition); ?, P ? 0.001 (compared with the Dlx1?2??? glutamate clonal
condition).
Before the stage of tangential cortical migration, individual MGE and LGE progenitors from the ventral forebrain but not the cerebral cortex give rise
Yung et al.
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subsequent cellular differentiation, they generated a large pro-
portion of multipotent clones composed of neurons, OLs, and
astrocytes (Fig. 2 A and C). By contrast, concurrent exposure of
these cells to Shh resulted in the preferential generation of
neuronal?OL clones at the expense of multipotent clones (Fig.
2 B and C). These observations suggest that the ventral gradient
morphogen, Shh, promotes neuronal?OL lineage restriction of
multipotent progenitors. Our studies have shown that before the
stage of tangential cortical migration of ventral forebrain (MGE
and LGE) progenitors, the cerebral cortex is incapable of
generating OLs and GABAergic neurons (see Fig. 1A). Ventral
forebrain progenitors are initially specified by the gradient
morphogen, Shh, and regional dorsal-ventral patterning is me-
diated by the dynamic interplay between different classes of
soluble signals (Shh and BMPs) and their downstream transcrip-
tional targets (12, 37). Therefore, we next investigated whether
exposure of premigratory stage cerebral cortical progenitors to
Shh could induce a ventral-to-dorsal fate switch that would now
allow these cells to give rise to GABAergic neurons and OL
species whose sequential elaboration could be potentiated by
changes in BMP signaling. Exposure of premigratory stage
cortical progenitors to Shh resulted in the dose-dependent
inhibition of the dorsal forebrain bHLH factor, Neurogenin1,
induction of the ventral forebrain GABAergic neuronal bHLH
factor, Mash1, and the OL homeodomain maturation factor,
Nkx2.2 (Fig. 2D) and concomitant up-regulation of the bHLH
GABAergic neuronal and OL lineage subtype factors, Olig2 and
Olig1 (not shown). This ventral-to-dorsal fate switch in progen-
itor cell lineage effectors was also associated with the Shh-
mediated dose-dependent elaboration of both GABAergic neu-
rons and OL species but not astrocytes (Fig. 2D).
Shh-Mediated Forebrain GABAergic Neuronal?OL Lineage Restriction
Requires Olig2 and Mash1. Previous studies have shown that Olig2
and Mash1 are expressed within the appropriate premigratory
stage ventral forebrain progenitor domains that are exposed to
endogenous levels of Shh signaling (21, 22, 25), and would
therefore represent the potential sites of initial GABAergic
neuronal?OL lineage restriction. In addition, these two bHLH
proteins, but not Olig1 or Nkx2.2, are selectively localized to
individual ventral forebrain progenitor species that give rise to
cortical GABAergic neurons and OLs but not to FGF-
responsive forebrain neural stem cells (S.-Y.Y., S.G., and
M.F.M., unpublished observations). Therefore, to more directly
examine the potential roles of Olig2 and Mash1 in Shh-mediated
GABAergic neuronal?OL lineage restriction, we performed
additional Olig2 and Mash1 gene manipulation paradigms.
When early embryonic dorsal forebrain progenitors underwent
clonal expansion in the presence of bFGF, they generated a large
proportion of clones containing glutamatergic neurons but not
clones containing GABAergic neurons and OL species (Fig. 3 A
and C). By contrast, clonal expansion of dorsal forebrain pro-
genitors in the presence of bFGF and Shh resulted in the
generation of clones containing both GABAergic neurons and
OL species as well as pure GABAergic neuronal and OL clones
(Fig. 3 B and D) at the expense of clones containing glutama-
tergic neurons (Fig. 3B). Ectopic expression of Olig2 at the time
of bFGF-mediated clonal expansion (Fig. 5A, which is published
as supporting information on the PNAS web site, www.pnas.org)
resulted in a conversion of the profile of differentiated clones to
a pattern more similar to that seen in the bFGF and Shh clonal
expansion condition (Fig. 3 A and B). Concurrent ablation of
Mash1 gene expression further reduced the total complement of
GABAergic neuronal?OL clones generated and altered the ratio
of pure GABAergic neuronal to OL clones without additional
changes in cellular viability (Fig. 3 A and C). Conversely, genetic
ablation of Olig2 in the presence or absence of simultaneous
inhibition of Mash1 gene expression (Fig. 5 B and C) at the time
of bFGF- and Shh-mediated clonal expansion resulted in the
conversion of the profile of differentiated clones to a pattern
similar to that seen in the bFGF clonal expansion condition (Fig.
3 A and B). In addition, ectopic production of astrocytes was only
evident in the combined Olig2 and Mash1 ablation condition
(not shown), suggesting that cooperative interactions between
Fig. 2.
forebrain stem cells and the selective induction of transcription factors in-
volved in GABAergic neuronal and OL lineage specification and maturation.
(A and B) Immunofluorescence micrographs of individual differentiated
clones derived from E12.5 cerebral cortical cells after clonal expansion under
control (bFGF, 10 ng?ml, A) and N-Shh (bFGF?N-Shh, 50 ng?ml, B) conditions
for 3 DIV, and subsequent plating as intact clonal populations under differ-
entiating conditions for an additional 6 DIV. Note that FGF-responsive clones
are multipotent (neurons: ?-tubulin, FITC; OLs: O4, cascade blue; astrocytes:
glial fibrillary acidic protein, tetramethylrhodamine B isothiocyanate),
whereasFGF?Shh-responsiveclonesarecomprisedofmorelineage-restricted
progeny (neurons: ?-tubulin, FITC; OLs:O4, tetramethylrhodamine B isothio-
cyanate). (C) Comparative clonal lineage profiles of bFGF- and bFGF?Shh-
responsive E12.5 cortical progenitor species plated and propagated under
cultureconditionsasnotedabove.Alldatapointsrepresenttheproportionof
target clones with its 95% confidence interval. Statistical analysis was calcu-
lated by using ?2analysis:*, P ? 0.001 (compared with individual clonal
lineage profiles in control condition). (D) Comparative proportion of individ-
ual clones containing Mash1, Neurogenin1, Nkx2.2, GABA, and O4 derived
from primary E12.5 cortical progenitors in the absence and presence of
increasingconcentrationsofShhafter6DIV.Statisticalanalysiswascalculated
by using ?2analysis: P ? 0.001 [for all data points relative to their control
(Ngn1), or Shh, 1 ng?ml (Mash1, Nkx2.2, GABA, O4) conditions].
Shh promotes neuronal?OL lineage restriction of early embryonic
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these bHLH factors are required to actively inhibit the astroglial
lineage during Shh-mediated neuronal?OL lineage restriction.
These observations suggest that both Olig2 and Mash1 are
required to promote Shh-mediated forebrain GABAergic neu-
ronal?OL lineage restriction and exhibit complementary and
cooperative factor interactions.
DevelopmentalModulationofBMPSignalingPromotestheSequential
Elaboration of Cortical GABAergic Neurons and OLs from Shh-Respon-
sive Forebrain Progenitors. We and other groups have shown that
BMP ligands inhibit the lineage specification but subsequently
potentiate the migration and differentiation of early-born glu-
tamatergic neurons from cortical VZ progenitors (28, 30, 32). By
contrast, we have also demonstrated that coincident with the
establishmentofthecorticalSVZ,BMPligandsnowenhancethe
specification of late-born cortical (GABAergic) neurons and
inhibit OL lineage elaboration (8, 32). Further, our studies have
revealed that the BMP antagonist, noggin, promotes the gener-
ation of OLs and retards further neuronal lineage elaboration (8,
30, 32). These overall observations suggested that dynamic
modulation of BMP signaling within the evolving cerebral cortex
may regulate the elaboration of GABAergic neurons and OLs
from MGE and LGE progenitor cells after tangential cortical
migration. We therefore evaluated the ability of individual
premigratory stage MGE and LGE progenitors to generate
GABAergic neurons and OL species in response to changes in
BMP signaling. Application of BMP2 to individual premigra-
tory-stage MGE and LGE progenitors enhanced the generation
of pure GABAergic neuronal clones at the expense of mixed
GABAergic neuronal?OL clones, whereas exposure to noggin
significantly enhanced the generation of pure OL clones also at
the expense of mixed GABAergic neuronal?OL clones (Fig. 4 A
and B). Our observations demonstrate that the actions of BMP2
are selective for GABAergic neurons (31). In addition, BMP
ligands from the BMP2?4 factor subgroup but not from three
additional BMP factor subgroups potentiated the elaboration of
GABAergic neurons (S.-Y.Y., S.G., and M.F.M., unpublished
observations). Different BMP ligands are expressed within the
Fig. 3.
GABAergic neuronal?OL species and glutamatergic neurons generated from E12.5 FGF-responsive cortical progenitors after clonal expansion (2 DIV) in the
absence or the presence of ectopic expression of Olig2 (Ad-O2) without or with concurrent ablation of Mash1 expression (Mash1as): Ad-GFP (adenoviral vector
expressing GFP alone), Ad-O2 (adenoviral vector expressing Olig2 and GFP), Mash1ss (Mash1 sense oligonucleotides), and Mash1as (Mash1 antisense
oligonucleotides). After clonal expansion in control and experimental conditions, intact clonal populations were plated under differentiating conditions for an
additional 6 DIV. All data points represent the proportion of target clones and its 95% confidence interval. Statistical significance was calculated by using ?2
analysis:*, P ? 0.001 (compared with GABA?OL clones in the bFGF?Ad-O2 clonal condition); ?, P ? 0.001 (compared with glutamatergic clones in the bFGF
condition). (B) The comparative proportion of clones containing GABAergic neuronal?OL species and glutamatergic neurons generated from E12.5 FGF?Shh-
responsive cortical progenitors in the absence or presence of ablation of Olig2 expression (Olig2as) without or with concurrent ablation of Mash1 expression
(Mash1as). O2ss, Olig2 sense oligonucleotides; O2as, Olig2 antisense oligonucleotides. Control and experimental conditions were subjected to clonal expansion
anddifferentiatingconditionsasdescribedabove.(C)ThecomparativeproportionofclonescontainingGABAergicneuronsorOLspeciesgeneratedunderclonal
cultureparadigmsidenticaltothosedescribedinA.Statisticalsignificancewascalculatedbyusing?2analysis:*,P?0.001(comparedwiththebFGF?Ad-O2GABA
clonal condition); ?, P ? 0.001 (compared with the bFGF?Ad-O2 O4 clonal condition). (D) The comparative proportion of clones containing GABAergic neurons
or OL species generated under clonal culture paradigms identical to those described in B.
Shh-mediated forebrain GABAergic neuronal?OL lineage restriction requires Olig2 and Mash1. (A) The comparative proportion of clones containing
Yung et al.
PNAS ?
December 10, 2002 ?
vol. 99 ?
no. 25 ?
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NEUROSCIENCE