1 Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel,
Switzerland
2 Novartis Institutes for Biomedical Research, Neuroscience, CH-4002 Basel,
Switzerland
3 Interdisciplinary Center for Neurosciences, University of Heidelberg, Im
Neuenheimer Feld 345, 69120 Heidelberg, Germany
* Author for correspondence (e-mail: yves.barde{at}unibas.ch)
Accepted 24 August 2004
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SUMMARY |
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Key words: Neural tube, Stem cells, Motoneuron, Radial glial cells, Neurotrophin receptors
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Introduction |
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In theory, there is no quantitative limit to cell replacement based on the
implantation of in vitro generated neural progenitors, and previous studies
have indicated that nestin-positive, ES-derived cells have the potential to
integrate in the host nervous system (e.g.
Brustle et al., 1997). However,
experiments of this kind have been typically performed with heterogeneous cell
populations (for a review, see Anderson,
2001
). We recently found that the addition of retinoic acid (RA)
to rapidly dividing mouse ES cells leads to the generation of a uniform
population of neural progenitors that display the characteristics of RG cells
found in the developing dorsal telencephalon
(Bibel et al., 2004
). This
finding offered the possibility to test the differentiation potential of a
homogenous cell population corresponding to progenitors participating in
normal brain development.
RG cells are the first cell type that can be distinguished from
neuroepithelial cells, and they have traditionally been considered to guide
the migration of newly born neurons and to subsequently become astrocytes (for
a review, see Rakic, 2003).
Recently, they were also discovered to generate neurons
(Malatesta et al., 2000
), and
it now appears that most pyramidal neurons in the developing telencephalon
derive from RG cells (Malatesta et al.,
2003
). In the present study, we implanted Pax6-positive RG cells
in place of a portion of the chick neural tube, and examined their fate
several days after implantation.
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Materials and methods |
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Cell culture
Mouse ES cells were deprived of mouse embryonic fibroblasts and cultured on
gelatine-coated dishes containing Dulbecco's Modified Eagle Medium and
leukemia inhibitory factor (LIF, 1000 U/ml) (for details, see
Bibel et al., 2004). To
facilitate their detection in the host, we used ES cells engineered to express
green fluorescent protein (GFP) from both tau alleles (for details,
see Tucker et al., 2001
;
Bibel et al., 2004
). Embryoid
bodies (EBs) were formed in bacteriological dishes for a period of 8 days,
with the addition of 5 µM all-trans RA (Sigma) during the last 4 days. In
some experiments (see Results), EBs were used 36 hours after the beginning of
their formation. EBs were fixed in 4% paraformaldehyde for 30 minutes,
incubated in 30% sucrose for 12 hours, embedded in cryomedium (OCT, Sakura)
and stored at 80°C until cryosectioning. In some experiments, 10
µM bromo-deoxyuridine (BrdU, Sigma) was added to EB cultures 3 hours prior
to fixation.
Chick embryo experiments
Fertilized chick eggs were incubated at 38.5°C and 80% humidity for
approximately 42 hours, until they reached the 19-21 somite stage. Embryos
were staged according to Hamburger and Hamilton
(Hamburger and Hamilton,
1951). Two millilitres of albumen was removed from the egg and a
portion of the upper eggshell was opened. To visualize the embryo, drawing ink
(Pelikan, A17) was dissolved in PBS (16 µl/ml) and injected under the
blastoderm. One neural fold was removed over a length of 4 somites, at the
level of the forelimb bud, by tearing the tissue with glass needles.
RA-treated EBs were incubated with trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA)
at 37°C for 10 minutes. EBs are typically heterogeneous in size and those
corresponding approximately to the size of the gap to be filled were selected
for the implantation experiments. RA-untreated EBs were trypsinized for 6
minutes. After incubation with trypsin, one EB was transferred, with a pipette
tip, onto the top of the missing portion of the neural tube and implanted
manually using a tungsten needle. By the end of these manipulations, the EB
had become a loose cell aggregate, which helped to accommodate it in the
appropriate position. Trypsin-treatment of EBs was found to be essential, as
untreated EBs remained compact and did not integrate into the host
environment. After sealing and incubation for 6 days, the embryos were removed
from the eggs, examined for GFP fluorescence and fixed in 4% paraformaldehyde
for 4 hours. Following incubation in 30% sucrose for 36 hours, they were
embedded in cryomedium and stored at 80°C for cryosectioning.
Immunohistochemistry
Sixteen micrometre thick cross-sections were rinsed in PBS and incubated
for 30 minutes in blocking solution containing 10% serum and 0.2% Triton in
PBS (7% Triton was used for Oct3/4 staining). Sections were then incubated
with primary antibodies in blocking solution for 12 hours at 4°C. The
following antibodies were used at the indicated dilutions: Isl1 (1:500, gift
from S. Arber, Biozentrum, University of Basel, Switzerland), Brn3a (1:10000,
gift from E. Turner, UCSD, USA), pan-Trk C-14 (1:1000, Santa Cruz), Glast
(1:1000, Chemicon), Oct3/4 N-19 (1:20000, Santa Cruz), Sox2 AB5770 (1:3000,
Chemicon), BrdU (1:1000, Sigma), and a p75 serum raised against the
bacterially expressed cytoplasmic domain of rat p75 (1:1000). The antibodies
40.3A4 (Isl1, 1:1500), 4F2 (Lim1/2, 1:500), 81.5 C10 (Mnr2, 1:500), Pax6
(1:1000), 74.5A5 (Nkx2.2, 1:50), Nestin (1:10), Rc2 (1:10), 50.5A5 (Lmx1,
1:50) and Pax7 (1:500) were obtained from the Developmental Studies Hybridoma
Bank maintained by the University of Iowa. In all cases, PBS was substituted
for the primary antibodies to test for unspecific labelling of secondary
antibodies. Sections were rinsed in PBS repeatedly and incubated with the
following antibodies for 1 hour at room temperature: anti-rabbit Rhodamine Red
X-conjugated antibody and anti-mouse Cy3 antibody (1:1000, Jackson),
anti-guinea pig antibody (1:1000, gift from S. Arber). Secondary antibodies
were combined with the nuclear stain Hoechst 33342 (10 µg/ml, Sigma).
Sections were rinsed in PBS and mounted. Sections used for BrdU staining were
previously incubated in 2 N HCl for 30 minutes at 37°C, then neutralized
in 0.1 M sodium tetraborate for 30 minutes and rinsed in PBS. Pictures were
collected with a Zeiss Axioplan2 Imaging fluorescent microscope and processed
with Adobe Photoshop 7.0.
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Results |
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Cells from RA-treated EBs colonize dorsal root ganglia but fail to differentiate into sensory neurons
In the chick embryo, neural crest cells delaminate from the dorsal neural
tube and start to migrate to the periphery to form the PNS at E2
(Le Douarin and Kalcheim,
1999). As our progenitors were implanted at these developmental
stages, we next examined whether donor cells could colonize the PNS.
GFP-positive cells were frequently found in the host DRG
(Fig. 4A and Tables
1,
2). However, unlike their chick
counterparts in the DRG, mouse neurons never expressed the transcription
factors Brn3a (Fig. 4B) and
Isl1 (Fig. 4C), which are
markers that define most neurons in that structure
(Anderson, 1999
). Surprisingly,
we never found mouse neurons elongating axons outside the DRG, even though GFP
expression indicated their neuronal identity. These cells expressed p75 at
high levels (Fig. 4D), but they
failed to express detectable levels of Trk receptors
(Fig. 4E).
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Discussion |
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Large numbers of EB-derived cells differentiate into neurons in the host
Six days after implantation, the vast majority of the progenitors were
found to differentiate in the spinal cord into Lim1/2+ interneurons and
Mnr2+/Isl1+ motoneurons. The latter extended axons towards the periphery and
expressed the neurotrophin receptors p75 and Trk, both in their somata and
axons. In addition to the identification of donor cells using GFP, we also
monitored, by nuclear staining, the fate of other cells that survived in the
chick embryo but failed to differentiate into neurons. Both in the spinal cord
and the DRG, about 80% of the mouse nuclei were found to belong to GFP+ cells.
A previous study has indicated that there is a complete overlap between cells
expressing GFP from the tau locus and those positive for the antibody
TuJ1 that recognizes a neuron-specific form of tubulin
(Tucker et al., 2001). In
addition, we also found that, in the spinal cord, many GFP-negative mouse
cells expressed either Lim1/2 or Mnr2 (data not shown), suggesting that they
were on their way to become post-mitotic interneurons or motoneurons,
respectively. Although it may seem surprising that our progenitor cells only
give rise to few cells not belonging to the neuronal lineage, our experiments
did not go beyond E8, which is before the time when large number of astrocytes
are generated in the spinal cord. It is possible that some of the GFP-negative
cells may later go on to differentiate into astrocytes and other cell types.
Our results also indicate that the implanted progenitors are able to respond
to patterning signals in the spinal cord, generating spinal cord interneurons
and motoneurons in a time- and position-dependent manner. At present, in vivo
cell lineage studies have not rigorously proven that Pax6-positive RG cells in
the spinal cord generate motoneurons and subtypes of interneurons, but this
appears quite likely. Indeed, the pattern of expression of RG markers and of
Pax6 in the spinal cord, as well as the decrease of motoneuron numbers in the
small eye mutant (Ericson et al.,
1997
), are compatible with this interpretation.
RA-treated EBs fail to elongate axons in the DRG
After implantation in the neural tube, the progenitors also exhibited a
migratory behaviour and colonized the adjacent DRG. Strikingly, while most
differentiated into GFP+ neurons in the host DRG, they failed to elongate
axons or to express Brn3a and Isl1, which define most neurons in that
structure. This is in contrast to donor-derived motoneurons in the spinal cord
that were Isl1+ and extended axons to the periphery, a result that does not
support an intrinsic limitation of the progenitors to express Isl1 or to
elongate axons. Moreover, while the mouse motoneurons expressed both p75 and
Trk receptors, donor neurons in the DRG did not express detectable levels of
Trk receptors. By contrast, these neurons expressed p75 at relatively high
levels. We previously showed that p75 intrinsically activates Rho and inhibits
axonal elongation (Yamashita et al.,
1999), and it is possible that p75 expression by donor neurons in
the DRG in the absence of detectable expression of Trk receptors may account
for their failure to elongate axons. During normal development, all sensory
neurons that express p75 also express at least one of the three types of Trk
receptor (Wright and Snider,
1995
). Our results suggest, then, that neurons located in the DRG
do not extend axons by default, even in the highly conducive environment
provided by developing DRG. Although we do not know why Trk receptors are not
expressed when mouse cells are located in the host DRG, we note that they fail
to express the POU transcription factor Brn3a. It has been shown that the
expression of all Trk receptors is compromised in the trigeminal ganglia of
Brn3a-/- mice, while the expression of p75 is not affected
(Huang et al., 1999
). In
addition, Brn3a has recently been shown to directly induce transcription of
TrkA in the DRG (Ma et al.,
2003
). Thus, the absence of Brn3a expression by donor neurons in
the DRG may be related to their failure to express Trk receptors. Why the
expression of Brn3a is not turned on is unclear, but we note that in the DRG,
the progenitors of Brn3a cells are not Pax6-positive. In view of these results
with the DRG, we performed similar implantation experiments with RA-untreated
ES cells. Virtually all ES cells at the time of implantation expressed Oct3/4,
one of the markers correlating with pluripotency of ES cells
(Niwa et al., 2000
;
Boiani et al., 2002
). We found
that these cells also survived in large numbers after implantation. Like
RA-treated cells, they also differentiated into Lim1/2+ interneurons, and into
Mnr2+ and Isl1+ motoneurons, extending long axons towards the periphery that
were positive both for Trk and p75 neurotrophin receptors. We observed,
however, that RA-untreated ES cells generated fewer motoneurons than
RA-treated cells, as judged by the expression of Mnr2 and Isl1. Recent studies
indicate that somite-derived RA plays an early role in the acquisition of a
neural fate by neural tube cells (Diez del
Corral et al., 2003
), and that the same molecule can further
promote these cells into a motoneuron differentiation pathway, even in the
absence of sonic hedgehog (Novitch et al.,
2003
). It is therefore conceivable that pre-treatment with RA may
not only induce neural differentiation of ES cells, but also brings these
cells closer to a motoneuron fate. ES cells also colonized the host DRG, but,
in contrast to RA-treated cells, they acquired expression of the markers Brn3a
and Isl1. These cells elongated axons outside the DRG both towards the spinal
cord and the periphery, and they expressed Trk receptors in addition to p75.
The proportion of ES cells colonizing DRG and differentiating into neurons in
that structure was similar to that observed for RA-treated cells. Thus, the
failure of RA-treated cells to express DRG markers and to elongate axons did
not result from a higher number of cells colonizing the DRG and
differentiating into neurons. We also note that neither RA-treated nor
RA-untreated cells colonizing the DRG ever expressed spinal cord markers.
Restricted developmental potential of RA-treated EBs
In vivo, RG cells expressing the markers Rc2, BLBP and Glast are widely
distributed throughout the embryonic CNS
(Kriegstein and Gotz, 2003).
However, not all of them are neurogenic. For example, RG cells in the
ganglionic eminence do not substantially contribute to the neuronal population
found in the striatum, or to the interneuron population in the cerebral
cortex, and the neurogenic potential of RG cells seems to correlate with their
expression of Pax6 (Heins et al.,
2002
; Malatesta et al.,
2003
). Thus, RG cells from the dorsal telencephalon express Pax6,
while RG cells located in the ventral telencephalon are Pax6-negative and are
essentially non-neurogenic. Recently, this conclusion was challenged by
Anthony et al. (Anthony et al.,
2004
), who suggested that RG cells may be neuronal progenitors in
most of the CNS. However, the BLBP promoter used by Anthony et al. to drive
the expression of Cre and to mark RG cell derivatives seemed to be effective
as early as E10.5, which may be before the time when BLBP is expressed in RG
cells. Fewer neurons are found in the cortex of Pax6 mutant mice, and
transfection of Pax6 into astrocytes seems to be sufficient to cause their
differentiation into neurons (Heins et
al., 2002
). The RA-treated ES cells used in our study have the
antigenic profile of RG cells found in the developing dorsal, but not in the
ventral telencephalon, as at the time of implantation essentially all of them
expressed Pax6. While previous work with these cells showed that, in vitro,
they differentiated into neurons with the characteristics of pyramidal cells
(Bibel et al., 2004
), we now
find that they can also respond to local cues, interpret them and
differentiate according to their position in the embryo. However, their
differentiation potential seems to be restricted. In particular, they cannot
acquire the typical antigenic and morphological features of peripheral sensory
neurons.
Conclusion
As neural progenitors with the characteristics of cortical RG cells can be
generated from ES cells in virtually unlimited amounts, they may represent a
useful source of defined cells to compensate for the loss of specific cell
types in the CNS, including motoneurons. It will be interesting to examine the
molecular determinants imposing developmental restrictions on such
progenitors, as this knowledge may become important in the context of specific
cell-replacement therapies.
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ACKNOWLEDGMENTS |
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