1 Developmental Genetics Program and the Department of Cell Biology, The
Skirball Institute of Biomolecular Medicine, New York University Medical
Center, 540 First Avenue, New York, NY 10016, USA
2 Center for Basic Neuroscience, UT Southwestern Medical Center, 5323 Harry
Hines Boulevard, Dallas, TX 75390-9111, USA
* Author for correspondence (e-mail: fishell{at}saturn.med.nyu.edu)
Accepted 10 August 2005
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SUMMARY |
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Key words: Cerebellum, Neural stem cell, Forebrain, Mouse
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Introduction |
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We are just starting to understand the functional role of newborn neurons
that are generated from adult NSC populations
(Kempermann et al., 2004;
Lledo et al., 2004
;
Schinder and Gage, 2004
;
Schmidt-Hieber et al., 2004
).
Despite the prevalence of NSCs in the adult CNS, the range of neuronal
subtypes that these cells generate in the adult brain seems to be limited
(Alvarez-Buylla et al., 2002
;
Doetsch, 2003a
). However, the
developmental potential of these populations appears to be broader when
challenged (Auerbach et al.,
2000
; Eriksson et al.,
2003
; Parmar et al.,
2003
; Suhonen et al.,
1996
). Indeed, when NSCs from the adult brain were introduced into
blastocysts they were shown to be capable of contributing to all germ layers,
albeit at very low frequency (Clarke and
Frisen, 2001
; D'Amour and
Gage, 2002
). Unfortunately, interpretation and generalization of
the data as a whole is difficult due to major differences in methodological
details used by different labs and the lack of markers to untangle the
complexity of neuronal subpopulations
(Bithell and Williams, 2005
;
Gage, 2000
;
Klein and Fishell, 2004
). A
direct comparison of the in-vitro and in-vivo potential of NSCs derived from
different regions of the CNS has not been systematically examined in a single
study.
Here we describe the existence and the potential of NSCs within the
embryonic and adult cerebellum, suggesting that despite the lack of evidence
for adult neurogenesis in the cerebellum
(Altman and Das, 1966), neural
stem cells may reside in the postnatal cerebellum. Notably, the cerebellum is
the only structure in the brain where the prevalent form of embryonic neural
progenitors (Anthony et al.,
2004
; Gaiano et al.,
2000
; Malatesta et al.,
2000
; Noctor et al.,
2001
), the radial glia cells, persists into adulthood. Having
identified a stem cell population in the cerebellum, we compare our findings
to NSCs from the ganglionic eminences of the forebrain, both in vitro and
after homotopic and heterotopic transplantation. We find that forebrain- and
cerebellar-derived neurospheres give rise to progeny in accordance with their
region of origin, but require local regional-specific cues to yield neurons
with the characteristics of region-specific subtypes.
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Materials and methods |
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The Math1EGFP mice were generated in a similar manner,
as previously described using the same Math1 enhancer element
(Lumpkin et al., 2003). By
contrast to Lumpkin and colleagues, the enhanced green fluorescent protein
(EGFP) expression matches the endogenous Math1 expression, and we do not see
any ectopic EGFP expression in the cortex and hippocampus.
Neurosphere cultures
The cerebellar anlage was identified and dissected by its position and
morphology (i.e. the dorsal portion of the metencephalon, superficial to the
pons and bordered posteriorly by the lumen of the fourth ventricle and
anteriorly by the mesencephalon). Whole cerebelli were prepared from P0 and
adult mice (>P42). The medial and lateral ganglionic eminences of the
telencephalon were dissected from Swiss Webster mice or
ß-actinEGFP E14.5 embryos, the meninges were removed and the
tissue chopped into smaller pieces and washed twice in cold DMEM. Embryonic
tissue was treated with 0.25% trypsin (Worthington) at 37°C for 5 minutes.
DNase I (0.1%; Sigma) and ovomucoid inhibitor (2 mg/ml ovomucoid, 0.1 g/ml BSA
in PBS) were then added and samples were triturated with a fire polished
Pasteur pipette. Postnatal tissue was dissociated using the papain
dissociation kit (Worthington) according to the manufacturer's instructions.
For the feeder layer, 1x105 cells/well from either the
cerebellum, the ganglionic eminences or E14 skin (i.e. fibroblasts) were
plated onto LabTekII CC2 chamber slides (Nunc) in DMEM/F12 with B27 supplement
and 2 mmol/l glutamine (Gibco) 3-5 days before the neurospheres were
seeded.
For the neurosphere assay, cells were plated at clonal density (1-2 cells/mm2) or FACsorted into 96-well plates and cultured in DMEM/F12 with B27 supplement, 2 mmol/l glutamine and 2 µg/ml heparin (Sigma) and EGF and/or FGF (20 ng/ml; Upstate Inc., Waltham, MA). The clonal nature of the neurosphere assays was confirmed under a microscope, which showed that all cells plated were single cell isolates or only one cell was plated per well.
To ascertain that the cells plated at low density gave rise to neurospheres that were clonally derived, we performed the following experiment. We mixed equal numbers of wild-type and GFP-expressing transgenic cerebellar progenitors and plated them at the density used in all our low-density neurosphere cultures (i.e. 1-2 cells/mm2). We reasoned that if re-aggregation of progenitor and differentiated cells was occurring the resulting neurospheres would be comprised of a mixture of both GFP-positive and -negative cells. Alternatively, if, as we hoped, the spheres were clonally derived, the resulting neurospheres would be either entirely GFP-positive or -negative. In a supplementary figure we show that, as hoped for, the latter proved true and in all cases neurospheres were either entirely GFP-positive or -negative, strongly supporting the clonality of the neurospheres used in our experiments (see Fig. S1B in the supplementary material). Notably this result was observed in all 100 neurospheres we examined.
After 7-10 days in culture, neurospheres were counted using a grid. The neurospheres were then transferred onto an age-matched feeder layer to induce differentiation. Although it did not change the qualitative outcome, neurospheres were generally placed on a feeder layer in DMEM/F12 supplemented with N2 and B27 and 2 mmol/l glutamate (Gibco) to facilitate the survival and differentiation of these cells (see Fig. S1C-F in the supplementary material). Cell cultures were analyzed after 5-10 days. Neurospheres grown at clonal density or clonally in 96-well plates were qualitatively indistinguishable in their developmental potential.
Immunocytochemistry, immunohistochemistry and quantification
Standard immunostaining procedures were used to stain the neurosphere
cultures and the histological sections. The following antibodies were used:
rabbit -AN2 (1:1000, gift from J. Trotter); rabbit
-calbindin
(1:5000; Swant), rabbit
-DARPP-32 (1:500; Chemicon), rabbit
-GABA (1:500; Sigma), rabbit
-GFAP (1:1500; Accurate, Westbury,
NY), chicken
-GFP (1:2000; Chemicon), rabbit
-glutamate (1:500;
Sigma), rabbit
-Math1 (1:100, provided by J. Johnson), mouse
-parvalbumin (1:500; Swant), mouse
-synaptophysin (1:200;
Sigma), mouse
-TAG1 (4D7; Developmental Studies Hybridoma Bank), mouse
-Tuj1/ßIIITubulin (1:1000; Covance). Secondary antibodies were
obtained from Jackson ImmunoResearch and were used at a dilution of 1:200.
Fluorescent images were obtained using an Axioscope (Zeiss), a cooled-CCD
camera (Princeton Scientific Instruments) and Metamorph software (Universal
Imaging). Confocal imaging was done on an LSM 510 Axioplan (Zeiss). Optical
sections were taken every 1 µm. Most of the presented confocal pictures are
single sections, but for some pictures two or three consecutive confocal
sections were combined after confirming the double labeling to better
illustrate the elaborate morphology of the transplanted cell.
For the quantification of the in-vitro double-labeled cells, pictures of five to ten visual fields of each condition were taken, and the total number of EGFP-expressing cells was counted to determine the percentage of these cells expressing specific markers.
For the quantification of the transplantation results in the forebrain, five regions (olfactory bulb, striatum, cortex, corpus callosum and hippocampus) per forebrain sections were chosen. The number of EGFP per region was determined and correlated with the total number of EGFP cells. For the quantification of the transplantation results in the cerebellum, the total number of EGFP-expressing cells was counted and correlated with the number of cells expressing specific markers.
Transplantations of neural stem cells
Neurospheres derived from EGFP-expressing transgenic mice were collected,
pooled and partially dissociated by triturating them at 37°C in
Leibowitz's L-15 medium (Gibco Invitrogene) with DNase I (0.1%; Sigma) and
collected by centrifugation. The cells were taken up in Leibowitz's L-medium
at a concentration of 105 cells/µl. The perinatal mouse
pups (P4) were cryo-anesthetized for 2 minutes. A small incision was made into
the skin overlaying the midbrain and the cerebellum for cerebellar injection
or the forebrain for forebrain injections using a surgical blade. The mouse
pup was placed in a self-made mold for stabilization and 1 µl of the cell
suspension was unilaterally injected into one cerebellar hemisphere or into
the subventricular zone (SVZ) with the help of a Hamilton syringe mounted
vertically into a stereotactic holder. Although the cell suspension was
injected slowly into the host brain, some leakage of some of the cell
suspension could not be avoided. The incision was sealed with Vetbond (World
Precision Instruments, Sarasota, FL), and the animals were then warmed to
36°C and returned to the litter. Twelve to 17 days after transplantation
the animals were either used for electrophysiology or perfused for
histological analyses.
Fluorescent activated cell sorting
Cerebellar anlagen of E14.5 Math1EGFP were dissected and
dissociated as described previously. The cell suspension was fluorescence
activated cell sorted (FACS) using a DakoCytomation MoFlo cell sorter. The
purity of sorted cells was determined by immunostaining for GFP 2 hours after
sorting. Math1EGFP-positive, -negative and unsorted fractions were
analyzed using the neurosphere assay.
Electrophysiology
GFP-positive and control GFP-negative profiles were recorded in the granule
cell layer in acute cerebellar slices obtained from neurosphere transplanted
mice (P15-P18) similar to that previously described
(D'Angelo et al., 1995;
D'Angelo et al., 1997
). In
brief, mice were anesthetized, killed by decapitation and the brain dissected
out and immediately immersed in ice cold Ringer's solution consisting of
(mmol/l): 125 NaCl, 2.5 KCl, 20 glucose, 25 NaHCO3, 1.25
NaH2PO4, 1 MgCl2 and 2 CaCl2 (pH
7.4 when bubbled with 95% O2, 5% CO2). Two hundred
micrometer slices were cut using Leica VT1000 vibratome and transferred to an
incubation chamber for a minimum of 1 hour prior to recording. Cells were
visualized using an infrared contrast system
(Stuart et al., 1993
). All
recordings were performed at room temperature. Intracellular electrodes were
pulled from borosilicate glass capillaries and filled with a solution
consisting of (mmol/l): 128 K-gluconate, 10 HEPES, 0.0001 CaCl2, 4
NaCl, 0.3 GTP, 5 ATP, 1 glucose (pH adjusted to pH 7.4 with KOH). Current
clamp recordings were performed using an Axoclamp 2B amplifier (Axon
Instruments, USA). Electrode capacitance was cancelled as best as possible
prior to obtaining electrical access to the cell. To ascertain if the cells
exhibited a current-voltage relationship consistent with them being cerebellar
granule cells, they were stimulated at 0.25 Hz with 300 or 500 millisecond
hyperpolarizing and depolarizing current pulses.
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Results |
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We compared the cerebellar cells with those derived from a combination of the medial and lateral ganglionic eminences of E14.5 forebrain. Although E14.5 cells from both the forebrain and cerebellum can generate neurospheres, twice the rate of neurosphere formation was observed in forebrain cultures (4.6±1.1%) than that of those derived from the cerebellum (2.1±0.2%). Furthermore, P0 and adult cerebellum maintained their ability to form neurospheres, albeit at reduced frequencies (1.3±0.1% and 0.3±0%: see Fig. S1A in the supplementary material). When dissociated and replated, neurospheres from each of these ages formed secondary neurospheres at the same rate as the primary neurospheres that gave rise to them (data not shown). Furthermore, when individual cerebellar cells were sorted into 96-well plates, neurospheres formed at higher frequency, as observed in low-density cultures (for adult tissue: 0.8±0.3% for 96-well plates and 0.3±0% for low-density cultures). Regardless of the culture conditions that led to their generation, the numbers and range of differentiated cell types generated from either embryonic or adult cerebellar-derived neurospheres was indistinguishable in vitro (see Fig. 1G-I in the supplementary material).
Embryonic and adult multipotent progenitor cells give rise to cerebellar-specific neuronal cell fates in vitro
Two of the advantages of studying the cerebellum are its relatively simple
cellular composition and the fact that the specific subclasses within this
structure are readily identifiable (Altman
and Bayer, 1996). Excluding the deep cerebellar nuclei, the
cerebellum is comprised of eight neuronal classes that can be distinguished by
morphology and immunocytochemistry as well as their location within the
cerebellar cortex.
The cerebellar neurons can be divided into the five `classic' neuronal
subtypes (granule cells, Golgi, stellate, basket neurons and Purkinje cells)
and into the less common neuronal subtypes (Lugaro, brush and candelabrum
neurons) (Flace et al., 2004;
Laine and Axelrad, 1994
).
The only excitatory neurons residing in the cerebellum are the granule
cells [except for the brush neurons, which are primarily located in the
granule cell layer of the flocculonodular node
(Dino et al., 1999)]. Granule
cells reside in the granule cell layer, have very small cell bodies, protrude
three to five dendrites and a long axon projecting into the molecular layer,
forming the parallel fibers. They can be identified with antibodies against
the excitatory neurotransmitter glutamate, and when in an immature state by
their expression of the cell surface protein TAG1
(Kuhar et al., 1993
;
Pickford et al., 1989
).
The remaining neuronal cell types are immunoreactive for GABA, the
inhibitory neurotransmitter used by these cells. Furthermore, all classical
interneuron classes within the cerebellum (Golgi, stellate and basket cells)
express the calcium-binding protein parvalbumin at high levels
(Bastianelli, 2003), whereas
Purkinje cells also express the calcium-binding protein calbindin
(Altman and Bayer, 1996
;
Celio, 1990
;
Rogers, 1989
). Basket and
stellate cells reside in the molecular layer, whereas Golgi cells are situated
in the granule cell layer. Most prominent are the Purkinje cells, which form a
discrete row of cells between the molecular and internal granule cell layers.
The Purkinje cells extend large dendritic arborizations into the molecular
layer and send their axon to the white matter track
(Altman and Bayer, 1996
).
Finally, the recently described candelabrum neurons
(Laine and Axelrad, 1994
) and
Lugaro neurons are located directly underneath the Purkinje cell layer. Lugaro
cells project their dendrites horizontally and remain within the molecular
layer (Laine and Axelrad,
2002
).
Using immunocytochemical markers, we analyzed the potential of cells within
cerebellar neurospheres, which upon removal of the growth factors and
presentation of an adhesive substrate undergo differentiation. The use of
neurospheres derived from a mouse line that ubiquitously expresses EGFP under
the ß-actin promoter (Okabe et al.,
1997) allowed neurosphere-derived cells (expressing EGFP) to be
distinguished from feeder-layer-derived cells.
Regardless of the age of the donor tissue, cerebellar neurospheres can give
rise to populations characteristic of each of the major cell subtypes observed
within the cerebellum. In all instances, a subset of cells derived from
embryonic (Fig. 1A-E) or adult
(Fig. 1F-K) cerebellar
neurospheres differentiated into GABA-ergic
(Fig. 1A,F; E14.5:
30.2±1.2%; adult: 26.3±2.6%) and parvalbumin-expressing neurons
(Fig. 1B,G; E14.5:
27±2.2%; adult: 17.3±2%). We also observed calbindin-expressing
neurons with the morphology of Purkinje cells, as indicated by their
elaborated dendritic arborization and the presence of a single long axon
(Fig. 1C,H; E14.5:
4.9±1.3%; adult 4.3±0.7%)
(Baptista et al., 1994).
Surprisingly, we were also able to generate a population of cells with the
morphology and the immunocytochemical profile of granule cells. These cells
possessed a small rounded cell body, had thin projections and expressed
glutamate (Fig. 1D,I; E14.5:
29.4±0.3%; adult: 29.4±3.2%) or TAG1
(Fig. 1E,J; E14.5:
29.4±1.3%; adult: 27.4±3.3%). The presence of this population
was unexpected, as the cerebellar GABA-ergic progenitor population is thought
to be segregated early in development (
E10.5) from the granule precursor
cells (Goldowitz and Hamre,
1998
; Wingate and Hatten,
1999
). In addition to the various cerebellar neuronal populations,
we also observed oligodendrocytes expressing the oligodendroglial precursor
marker AN2/NG2 and astrocytes, expressing the intermediate filament GFAP (data
not shown). Neurosphere-derived neurons intermingle with feeder-layer-derived
cells and express the synaptic vesicle marker synaptophysin
(Fig. 1K), suggesting that
these neurons have the ability to generate synaptic vesicles in vitro. All
neurospheres contained cells with the immunological characteristics of granule
cells, interneurons and astrocytes, suggesting that the cerebellar stem cell
population we are studying may be uniform in their potency, at least under
in-vitro conditions. However, only a subpopulation of neurospheres contained
cells with the characteristics of Purkinje neurons. While this may suggest
that only a subpopulation of cerebellar neural stem cells can generate this
cell type, it is also possible that this is simply the result of the low
frequency at which this cell type is generated.
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At E14.5, EGFP is expressed in the external granule layer of the cerebellum
(Fig. 2A). We dissected the
cerebellar anlagen of E14.5 Math1EGFP-expressing mice and used FACS
to separate EGFP-expressing cells (i.e. granule cell precursors,
8.8±0.1% of the whole fraction) from the cells that do not express EGFP
(78.4±3.9% of the remaining cerebellar cells;
Fig. 2B). Both fractions, as
well as an unsorted control population, were examined using the neurosphere
assay. Whereas the unsorted and the Math1/EGFP-negative cells formed
neurospheres at the previously observed frequency (unsorted: 2.3±0.2%,
Math1/EGFP-negative: 2.3±0.1%), the Math1/EGFP-positive cells failed to
form neurospheres (0.04±0.01%; Fig.
2C). This latter result could perhaps have been anticipated, as
previous work has demonstrated that granule cell proliferation is maintained
by sonic hedgehog (Dahmane and
Ruiz-i-Altaba, 1999;
Wechsler-Reya and Scott,
1999
).
The neurospheres derived from the
Math1/EGFP-negative fraction were then placed onto a cerebellar feeder-cell layer and allowed to initiate differentiation. After 2-3 days we observed the upregulation of the Math1EGFP expression within the cultures (Fig. 2D,E). This is consistent with the presence of granule cell precursors within these cultures, although based on this experiment we cannot rule out the possibility of the generation of other dorsally derived Math1-expressing cell types. FACS analysis of the whole culture (feeder-layer and differentiated neurospheres) confirmed that the cells were viable and expressed EGFP (Fig. 2D). Notably all EGFP expression was lost in these cultures after 1 week, suggesting that the granule cell precursors progressed to a Math1-negative differentiated state.
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Cerebellar- and forebrain-derived NSCs give rise to region-specific cellular phenotypes in vivo
To further analyze the differentiation potential of forebrain- and
cerebellar-derived neurospheres, we transplanted them in to the SVZ of
developing forebrain (postnatal day 4). As with our in-vitro experiments, the
transplanted cells could be unambiguously identified by their constitutive
expression of EGFP (Okabe et al.,
1997). Two weeks post-transplantation, the distribution,
morphology and expression of neural marker genes of transplanted cells were
examined. Cells from forebrain-derived neurospheres integrated into the host
tissue and migrated away from the injection site in a manner resembling the
normal migration of SVZ-derived neurons
(Fig. 3). Most EGFP-labeled
cells were found in either the rostral migratory stream (RMS) and olfactory
bulb (46.4±6.9%) or the cortex (36.6±5.6%). Fewer numbers of
cells were located in the striatum (6.5±3.5%), corpus callosum
(3.1±1.3%) and hippocampus (10.2±4.4%). Cells in the olfactory
bulb were mainly found in the inner granule cell layer of the olfactory bulb,
a subpopulation of which was immunopositive for GABA
(Fig. 3B1,B2). Cells within the
olfactory stream appeared to be migrating in chains and expressed the neuronal
progenitor marker Tuj-1 (Fig.
3C1,C2) (Doetsch and
Alvarez-Buylla, 1996
). Forebrain-derived cells were found
scattered in most layers of the cortex and were immunopositive for GABA,
suggesting that they are interneurons (Fig.
3D1,D2). Most cells that remained in the injection site expressed
the astroglial marker GFAP (Fig.
3E1,E2).
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We also did the converse experiment of transplanting both forebrain- and cerebellar-derived neurospheres into the cerebellum. Forebrain-derived cells integrated into the host tissue and could be found in all cerebellar layers, including the white matter. Virtually all forebrain-derived cells observed after a 2-week survival period appeared to be glial cells that expressed GFAP. Consistent with this neurospherederived forebrain, cells generally adopted the morphology of either astrocytes or Bergman radial glia (Fig. 5A-F) (only one neurofilament-expressing cell was found in the analysis of 16 brains; Fig. 5G-I). In no cases did we observe forebrain cells that acquired the morphologies or markers characteristic of cerebellar neurons (data not shown).
We also transplanted E14.5- or adult-derived cerebellar neurospheres into P4 cerebellum. Two weeks post-grafting, EGFP-expressing cells were found distributed throughout all the layers of the cerebellum, as well as in the white matter tracks. GABA-expressing neurosphere-derived cells were found located in both the molecular and the inner granule cell layer (E14.5: 20.4±5.4%; adult: 20.7±3.7%; Fig. 6AF). A few calbindin-expressing cells with the morphology of Purkinje cells were observed within the Purkinje cell layer (Fig. 6G-I), albeit only when derived from embryonic neurospheres and then with the same low frequency observed in vitro (0.5±0.4%). By contrast, transplantations of adult neurospheres, although they did not produce cells resembling Purkinje cells, gave rise to a population of cells that assumed the position and characteristic morphology of Lugaro cells (Fig. 6J-L). Notably, after either transplantation of embryonic or adult-derived cerebellar neurospheres the most abundant cell population observed possessed small cell bodies and were found in the inner granule cell layer (E14.5, 45.8±5.4%; adult, 58.9±5.3%). Consistent with this population being granule cells, they often both protruded 3-5 dendrites and were immunopositive for glutamate (Fig. 6M-R). In addition, subpopulations of cells after embryonic or adult neurosphere transplantation gave rise to glial cells. Notably, three-dimensional reconstructions of cells of each of these classes indicated that they were not multinuclear, suggesting that they do not result from cell fusion events.
To further characterize the cerebellar-derived population resembling
granule cells, we undertook an analysis of their electrophysiological
properties. From animals receiving neurosphere transplants, we recorded 36 of
the cells found in the internal granule cell layer of the cerebellum, using
whole cell patch clamp recording (EGFP-positive: 12 E14.5 NSCs, nine adult
NSCs and 15 EGFP-negative). The current-voltage response of the recorded cells
was ascertained from holding potentials of approximately 70 mV and
three classes were defined, non-spiking, immature and mature (see
Table 1), in both the
EGFP-positive and negative populations similar to that previously described in
young rats (D'Angelo et al.,
1997). Immature cells exhibited long-duration, non-repetitive
intermediate- and high-threshold calcium spikes
(Fig. 6S,U)
(D'Angelo et al., 1997
).
Mature granule cells of all populations exhibited characteristic inward
rectification at subthreshold voltage steps and repetitive fast spikes (spike
half width
2 mseconds) (Fig.
6T,V). The ability of the EGFP-positive cells to generate action
potentials and their overall passive membrane properties, size and position
are strongly indicative of a cerebellar granule cell phenotype. In addition,
we observed spontaneous excitatory synaptic potentials when recording from
grafted cells [sensitive to the glutamatergic antagonist CNQX (10-20
µmol/l; n=3)], demonstrating that they receive afferent input.
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Discussion |
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Cerebellar stem cells exist in both the embryonic and adult cerebellum
In this study we extend previous analysis of CNS stem cells by
characterizing their existence in the embryonic and adult cerebellum. While
the existence of such a population in the embryonic and adult telencephalon is
well recognized (Alvarez-Buylla and
Garcia-Verdugo, 2002; Doetsch,
2003b
), the presence of these populations in other parts of the
nervous system is still a matter of debate
(Gould et al., 1999
;
Horner et al., 2000
;
Kornack and Rakic, 2001
;
Magavi et al., 2000
;
Zhao et al., 2003
). Similarly,
the majority of NSC data suggest that they generically give rise to all cells
in the CNS. Such claims are compromised by the lack of analysis of the
regional character of these cells by markers other than ubiquitous markers of
neurons and glia. The very existence of this population in the cerebellum
raises the question of whether some basal neurogenesis may occur in the adult
cerebellum.
|
|
Forebrain and cerebellar NSCs maintain their regional character
Having demonstrated the existence of cerebellar stem cells, we sought to
compare their differentiation potential to those stem cell populations known
to exist in the forebrain. Indications that NSCs retain discrete regional
character in vitro have come from a number of recent studies
(Hitoshi et al., 2002;
Horiguchi et al., 2004
;
Jensen et al., 2004
;
Parmar et al., 2002
). These
studies suggest that the NSCs from distinct regions of the brain maintain some
aspects of their molecular profile and can differentiate into some but not all
neuronal classes in accordance with their regions of origin
(Hitoshi et al., 2002
;
Horiguchi et al., 2004
;
Parmar et al., 2002
). However,
extrinsic factors appear to influence the developmental potential of NSCs by
promoting their intrinsic cellular program when the NSCs are placed in a
matching environment (Hitoshi et al.,
2002
; Jensen et al.,
2004
). Here we present the first systematic comparison of NSCs
derived from two different regions of the CNS. Our in-vitro results support
this trend by showing that after differentiation both forebrain and cerebellar
NSCs give rise to neuronal cell types appropriate to their region of
origin.
Furthermore, as shown by other researchers
(Auerbach et al., 2000;
Gage et al., 1995
;
Parmar et al., 2003
;
Sabate et al., 1995
;
Suhonen et al., 1996
), our
transplantation studies show that forebrain-derived NSCs integrate when
grafted into the neonatal SVZ, make use of normal migratory routes and
differentiate appropriately. Moreover, forebrain-derived NSCs only give rise
to astrocytes when transplanted to the cerebellum as previously described
(Suhonen et al., 1996
). By
contrast, cerebellar derived-NSCs can integrate and differentiate into
cerebellar neurons when transplanted into the developing cerebellum (see
below). Conversely, when cerebellar-derived NSCs are grafted into the SVZ they
fail to migrate along the RMS and largely differentiate into astrocytes.
These observations suggest that NSCs, as well as the populations they give
rise to, retain regional character that reflects their place of origin. Upon
passaging, numerous examples suggest that to varying degrees region
characteristics are lost (Gabay et al.,
2003; Hack et al.,
2004
; Hitoshi et al.,
2002
; Jensen et al.,
2004
; Ostenfeld et al.,
2002
; Parmar et al.,
2002
; Santa-Olalla et al.,
2003
). In this regard, our studies only examine neural progenitors
expanded in culture without passaging, and even so full differentiation to
normal fates could only be achieved in vivo upon homotopic
transplantation.
Considerable data support the notion that NSCs in the CNS are maintained by
specialized niche environments
(Alvarez-Buylla and Lim, 2004;
Doetsch, 2003b
;
Doetsch et al., 1999
;
Doetsch et al., 1997
;
Lai et al., 2003
;
Lim et al., 2000
;
Machold et al., 2003
). The
progressive loss of regional character of NSCs when expanded through multiple
passages in vivo probably reflects the requirement of these specialized niches
in reinforcing the regional character of NSCs. However, our observation that
NSCs expanded in vitro without passaging fail to attain appropriate regional
character when transplanted heterotopically supports the argument that
epigenetic cues in their normal postmitotic environment are required for NSCs
to express proper regional character in vivo.
Studies examining the fate of heterotopically transplanted NSCs have
generated mixed results. Work by the Gage laboratory has shown that
hippocampal NSCs transplanted into the olfactory bulb can adopt seemingly
normal olfactory granule cell identity
(Auerbach et al., 2000;
Suhonen et al., 1996
). By
contrast, heterotopic transplantation of NSCs from the spinal cord to the
forebrain has at best led to NSCs adopting phenotypes only partially
appropriate to the host region
(Shihabuddin et al., 2000
;
Yang et al., 2000
). These
results are generally consistent with our findings that both forebrain and
cerebellar NSCs require homotypic environments to differentiate with
appropriate regional character. The exception to this rule appears to be when
the transplants are restricted to the same region of the neuraxis
(Auerbach et al., 2000
;
Suhonen et al., 1996
).
Cerebellar stem cells can functionally integrate into the perinatal cerebellum
When transplanted into the perinatal cerebellum, embryonic as well as adult
cerebellar-derived neurospheres generated cells with characteristics of
multiple neuronal and glial cerebellar cell types. Both embryonic and adult
NSCs were observed to give rise to neurons that resemble GABA-ergic
interneurons, granule cells, oligodendrocytes and astroglia. In addition, at
low frequencies transplanted embryonic cerebellar NSCs gave rise to Purkinje
cells, while adult cerebellar NSCs generated cells that resemble Lugaro
neurons, indicating that the developmental potential of neurosphere-derived
NSCs might get restricted over time, as has been shown for acutely dissociated
cells grafted into the developing cerebellum
(Carletti et al., 2002). These
findings are consistent with recent evidence that demonstrates that the
potential of neural crest stem cells changes during maturation
(White et al., 2001
).
Alternatively, the populations of NSCs we isolated from E14.5 cerebellum
versus the adult cerebellum may represent distinct populations. Given that
these two populations were indistinguishable based on their in-vitro
differentiation potential and only differed in their generation after in-vivo
grafting of neurons with Purkinje (E14.5) versus Lugaro characteristics
(adult), we think it likely that they represent the same population, the
potential of which changes somewhat over time. This is consistent with data
suggesting that the embryonic radial glial progenitor population in the
forebrain ultimately transforms into the adult NSC `B cell' population
(Merkle et al., 2004
).
Patch-clamp recordings of NSC-derived neurons revealed that those resembling granule cells acquired electrophysiological properties that were indistinguishable from the host cerebellar granule cell population. The functional integration of these cells into the host environment was indicated by the frequent occurrences of spontaneous excitatory postsynaptic potentials, demonstrating that the grafted cells received afferent input.
There is the notion that the in-vivo acquisition of neural identities can
be attained through cell fusion
(Alvarez-Dolado et al., 2003).
If this phenomenon explained our findings, it is surprising that
cerebellar-derived NSCs could give rise to the neurons resembling the
different normal cerebellar populations, while forebrain cells do not.
Similarly, reports of in-vivo cell fusion suggest that this phenomenon
apparently occurs at low frequencies. It is implausible that the large number
of integrated cells in the cerebellum of animals receiving neurosphere grafts
could be accounted for by this phenomenon. Taken together, our data support
the idea that NSCs persist in the cerebellum into adulthood. Furthermore, our
analysis suggests that NSCs derived from different brain regions possess
intrinsic developmental character.
The fact that grafted cerebellar stem cells can assume appropriate
cytoarchitecture in the perinatal cerebellum suggests that cues permitting the
proper integration of nascent cells persist at least during this phase of
development. These cues are likely to be permissive rather than instructive,
as forebrain-derived stem cells do not adopt cerebellar neuronal character
when transplanted into the neonatal cerebellum. It would be interesting to see
whether cerebellar NSCs cells can also integrate into more mature cerebellum.
Similarly, it would be interesting to ascertain how precisely various
cerebellar neuronal populations can be generated from cerebellar neurospheres.
In this regard, recent work has unexpectedly demonstrated that by E13.5 three
distinct sublineages of granule cells exist
(Zong et al., 2005). As more
understanding of the lineages and diversity of cerebellar cells is garnered,
it will be intriguing to explore more fully the differentiation capacity of
both embryonic and adult cerebellar NSCs.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/20/4497/DC1
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