1 Departments of Internal Medicine and Cell and Developmental Biology, 1500 East
Medical Center Drive, University of Michigan, Ann Arbor, MI 48109-0934,
USA
2 Division of Biology 216-76, California Institute of Technology, Pasadena, CA
91125, USA
3 Department of Cell Biology, Erasmus University Medical Center, 3000DR
Rotterdam, The Netherlands
4 The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037,
USA
* Howard Hughes Medical Institute
Author for correspondence (e-mail:
seanjm{at}umich.edu)
Accepted 3 September 2004
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SUMMARY |
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Key words: Neural crest stem cell, Peripheral nerve development, Fate-mapping
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Introduction |
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Circumstantial evidence suggests that NCSCs give rise to more than just
Schwann cells in developing nerves. Culture of single cells from the
developing sciatic nerve yields several different types of colonies, including
multilineage NCSC colonies (containing neurons, Schwann cells and
myofibroblasts), colonies that contain only Schwann cells and myofibroblasts,
colonies that contain only Schwann cells, and colonies that contain only
myofibroblasts (Morrison et al.,
1999). This raises the possibility that NCSCs give rise to
restricted progenitors within the nerve, which in turn differentiate into
Schwann cells and fibroblasts. If single sciatic nerve NCSCs are subcloned
after proliferating in culture they generate additional NCSCs, as well as
colonies that contain only Schwann cells and myofibroblasts, colonies that
contain only Schwann cells, and colonies that contain only myofibroblasts
(Morrison et al., 1999
). Nerve
NCSCs may undergo progressive restrictions in vitro and in vivo to form both
Schwann cells and fibroblasts.
Recent fate-mapping studies in vivo have raised questions about whether the
fate of neural progenitors in vivo can be accurately predicted based on their
function in culture (Gabay et al.,
2003). Because culture conditions often dysregulate progenitor
patterning in an unphysiological way
(Anderson, 2001
), neural
progenitors might readily generate cell types in culture that they would never
generate in vivo. In this regard, the ability of nerve (and other trunk) NCSCs
to generate myofibroblasts in culture
(Morrison et al., 1999
)
appeared to contrast with the failure to observe a contribution of trunk
neural crest cells to fibroblast-type derivatives in vivo
(Le Douarin, 1982
). This
raises the question of whether the potential of nerve NCSCs to make
fibroblasts in culture is ever expressed in vivo. More generally, it is
important to begin to assess the fate of neural stem cell populations during
normal development, in addition to examining the developmental potential of
these cells in culture.
If NCSCs differentiate into both Schwann cells and fibroblasts within
nerves, what is the ultimate fate of the fibroblasts? There are a variety of
cell types present within peripheral nerves in addition to Schwann cells,
including perineurial cells, pericytes and endoneurial fibroblasts
(Fig. 1). The embryonic origins
of perineurial cells, pericytes and endoneurial fibroblasts are uncertain and
controversial. Based on morphological criteria, some investigators have argued
that perineurial cells are neural crest derived
(Hirose et al., 1986), while
others have argued they are not (Low,
1976
). The observation that embryonic fibroblasts from the cranial
periosteum could form perineurial cells in culture supported a mesodermal
origin for these cells (Bunge et al.,
1989
). Although pericytes have been presumed to be mesodermally
derived, the vascular smooth muscle around the cardiac outflow tracts is
neural crest derived (Jiang et al.,
2000
; Kirby and Waldo,
1995
), and so some vascular smooth muscle cells in other locations
might also be neural crest derived. Little is known about the properties of
endoneurial fibroblasts or their embryonic origin.
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Materials and methods |
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Tissue preparation for light and electron microscopy
P11 mice were anesthetized with ketamine/xylazine and perfused through the
heart with 0.25% glutaraldehyde in D-PBS. Sciatic nerves were dissected and
fixed for an additional 15-20 minutes in 0.25% glutaraldehyde. Nerves were
then rinsed twice in PBS and stained in either 2 mM X-gal (for light
microscopy) or 2 mM 5-bromo-3-indolyl-ß-D-galactoside (bluo-gal;
Invitrogen Life Technologies, Carlsbad CA, USA; for electron microscopy).
X-gal and bluo-gal were dissolved in D-PBS with 20 mM potassium ferrocyanide,
20 mM potassium ferricyanide, 2 mM MgCl2 and 0.3% Triton-X, as
previously described (Weis et al.,
1991). The staining was done for 6-10 hours at 37°C. For light
microscopy, nerves were removed from the X-gal buffer, rinsed in PBS,
postfixed in 4% paraformaldehyde for 1 hour, cryoprotected in 15% sucrose, and
embedded in gelatin. Frozen sections were cut on a Leica 3050S cryostat. For
electron microscopy, nerves were removed from the bluo-gal buffer, rinsed in
PBS, then postfixed in 2.5% glutaraldehyde overnight at 4°C. Nerves were
then rinsed in 0.1 M sodium phosphate buffer and refixed in 1% OsO4
(Electron Microscopy Sciences, Fort Washington PA, USA) for 1 hour at room
temperature. The nerves were rinsed in sodium phosphate buffer then dehydrated
in an ethanol series (30%, 50%, 70%, 95% and 100% ethanol) before infiltrating
and embedding in Spurr resin (Bozzola and
Russell, 1992
). Semithin sections (1 µm) were cut with a glass
knife. Thin sections (70 nm) were cut with a diamond knife and examined
without further staining to facilitate visualization of the bluo-gal crystals.
Some sections were stained with uranyl acetate to reveal the basal lamina.
Note that the presence of the Triton-X detergent in the bluo-gal buffer
resulted in poor preservation of myelin sheaths. However, the Triton-X was
required for effective bluo-gal staining.
Immunohistochemistry for confocal microscopy
Sciatic nerves were fixed in 4% paraformaldehyde, cryoprotected, and
embedded in gelatin. Cross-sections of P11 sciatic nerves were cryosectioned
at 10-12 µm thickness, collected on pre-cleaned superfrost slides (Fisher
Scientific, Chicago IL, USA), dried for 1-2 hours and stored at
20°C. Slides were allowed to thaw, then were washed in PBS for 5
minutes, postfixed in 4% paraformaldehyde for 10 minutes, rinsed for
3x10 minutes in PBS, and blocked in 10% goat serum/0.2% Triton-X in PBS
for 1 hour. Slides were incubated in primary antibodies in blocking solution
overnight at 4°C. We used rabbit anti-ß-galactosidase (1:1000;
5'-3', Boulder CO, USA), with anti-smooth muscle action (1:200;
Sigma, St Louis MO, USA) and anti-PECAM (1:300; Pharmingen, San Diego CA,
USA), or anti-S100ß (1:1000; Sigma). Slides were washed for 4x15
minutes in 2% goat serum/0.2% Triton-X in PBS then incubated with secondary
antibodies diluted 1:200 in blocking solution for 1 hour at room temperature
in the dark. Slides were washed for 4x15 minutes in the dark, then
mounted in Prolong anti-fade (Molecular Probes) and analyzed on a Zeiss 510
laser confocal microscope.
Cell culture
Timed pregnant Sprague-Dawley rats were obtained from Charles River
(Wilmington MA, USA). E14.5 rat gut and sciatic nerve NCSCs were isolated by
flow cytometry and cultured under standard conditions, as previously described
(Bixby et al., 2002). Briefly,
dissociated cells from the sciatic nerve and gut were stained with antibodies
against the neurotrophin receptor p75 (192Ig) and
4 integrin
(Becton-Dickinson, San Jose CA, USA). The
p75+
4+ population was sorted into
`standard medium' at clonal density. The standard culture medium contained
DMEM-low (Gibco, product 11885-084, Grand Island, NY, USA) with 15% chick
embryo extract (Stemple and Anderson,
1992
), 20 ng/ml recombinant human bFGF (R&D Systems,
Minneapolis MN, USA), 1% N2 supplement (Gibco), 2% B27 supplement (Gibco), 50
µM 2-mercaptoethanol, 35 mg/ml (110 nM) retinoic acid (Sigma),
penicillin/streptomycin (Biowhittaker, Walkersville, MD, USA) and 20 ng/ml
IGF1 (R&D Systems). Some cultures were supplemented with 50 ng/ml Bmp4
(R&D Systems), 65 ng/ml Nrg1 (gift of CeNeS Pharmaceuticals, Norwood MA,
USA), and Delta-Fc or Fc. Note that Delta-Fc and Fc were prepared and added to
culture as described previously (Morrison
et al., 2000b
). All cultures were maintained in gas-tight chambers
(Billups-Rothenberg, Del Mar CA, USA) containing decreased oxygen levels, as
previously described, to enhance the survival of NCSCs
(Morrison et al., 2000a
).
After 6 or 14 days, cultures were fixed in acid ethanol (5% glacial acetic
acid in 100% ethanol) for 20 minutes at 20°C, washed, blocked and
triply labeled for peripherin (Chemicon AB1530; Temecula CA, USA), GFAP (Sigma
G-3893) and alpha SMA (Sigma A-2547), as described
(Shah et al., 1996
).
E13.5 mouse sciatic nerves were dissected and dissociated from timed pregnant transgenic mice. Unfractionated cells were plated at clonal density and cultured under standard conditions. The medium for the culture of mouse cells was the same as the medium used for rat cells except that 3:5 mixture of Neurobasal(Gibco):DMEM was used instead of DMEM. Cultures were fixed with 0.25% glutaraldehyde for 5 minutes and stained with X-gal.
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Results |
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Perineurial cells are not neural crest derived
To explore the fates of neural crest cells in the developing nerve, we
first examined whether perineurial cells in
Wnt1-Cre+loxpRosa+ pups expressed ß-gal.
Perineurial cells separate distinct bundles of nerve fibers within a nerve,
and regulate diffusion and cell movement between the epineurial and
endoneurial environments (Peters et al.,
1976) (Fig. 1). We
examined whether perineurial cells express ß-gal in sciatic nerves by
X-gal staining, immunohistochemistry and electron microscopy. In each case
there was a distinct absence of ß-gal expression in the perineurium
(Fig. 2), despite the fact that
perineurial cells readily expressed ß-gal in positive control nerves from
Rosa26 pups (see Fig. S3 in supplementary material) and
CMV-Cre+loxpRosa+ pups (data not shown). Upon examining
transverse sections through bluo-gal-stained nerves by electron microscopy
(Weis et al., 1991
), we rarely
observed any bluo-gal staining within perineurial cells (see below), despite
observing bluo-gal staining in the majority of Schwann cells within the same
sections (Fig. 2C;
Table 1).
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Pericytes and endothelial cells are not neural crest derived
To assess whether the pericytes and endothelial cells that are present
around small blood vessels in the endoneurium
(Fig. 1) were neural crest
derived, we performed immunohistochemistry using antibodies against the
endothelial marker PECAM1, the pericyte marker SMA and ß-gal in sciatic
nerve sections from Wnt1-Cre+loxpRosa+ pups. Neither
PECAM1+ endothelial cells nor SMA+ pericytes co-labeled
with antibodies against ß-gal, despite widespread ß-gal expression
by other cells within the nerve (Fig.
3). Pericytes expressed ß-gal in nerve sections from positive
control Rosa26 pups (see Fig. S3 in supplementary material), and endothelial
cells from Rosa26 mice widely express ß-gal
(Jackson et al., 2001). This
indicates that pericytes and endothelial cells within the sciatic nerve are
not neural crest derived.
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NCSCs undergo multilineage differentiation within the nerve environment in vivo
The data above raise the possibility that NCSCs differentiate into Schwann
cells and endoneurial fibroblasts after migrating into the developing nerve.
However, it remains possible based on the in vivo fate mapping experiments
that endoneurial fibroblasts arise from a neural crest lineage that is
independent of NCSCs, or that segregates prior to their migration into the
nerve. To address these possibilities, we examined the fates of desert
hedgehog (Dhh)-Cre expressing neural crest progenitors in vivo.
Dhh is not expressed by migrating neural crest cells, or by neural
crest progenitors that colonize ganglia, but is expressed in neural crest
progenitors within developing peripheral nerves
(Bitgood and McMahon, 1995;
Jaegle et al., 2003
;
Parmantier et al., 1999
). The
Bluo-gal-staining pattern in Dhh-Cre+loxpRosa+ fetuses
was consistent with the expected Dhh expression pattern, labeling
only developing nerves (Jaegle et al.,
2003
) and not migrating neural crest cells (see Fig. S5 in
supplementary material). We examined Bluo-gal-stained sections through the
sciatic nerves of P11 Dhh-Cre+loxpRosa+ mice and
littermate controls by electron microscopy
(Fig. 5). Within postnatal
nerves, we observed Bluo-gal staining in 60% of endoneurial fibroblasts and
63-67% of Schwann cells (Fig.
5A-D). Only 2.4% of perineurial cells appeared to label with
Bluo-gal. These data indicate that Dhh-expressing neural crest
progenitors within the nerve give rise to both Schwann cells and endoneurial
fibroblasts, in remarkably similar proportions.
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Common expression of Thy1 suggests a link between myofibroblasts in culture and endoneurial fibroblasts in vivo
The above experiments demonstrated that a small percentage of
myofibroblast-committed progenitors that can be cultured from sciatic nerve
are neural crest derived, and that endoneurial fibroblasts arise from the
neural crest in vivo. These observations suggested that neural crest-derived
cells that differentiate in vitro as myofibroblasts were fated to form
endoneurial fibroblasts in vivo. However, the marker that we have used to
identify myofibroblasts in culture, SMA
(Fig. 7), is not expressed by
endoneurial fibroblasts in vivo. Thus, we wondered whether myofibroblasts are
capable of expressing markers of endoneurial fibroblasts. All myofibroblasts
in culture expressed fibronectin, collagen and vimentin (data not shown),
which are also expressed by endoneurial fibroblasts in vivo
(Jaakkola et al., 1989;
Peltonen et al., 1987
). A more
selective marker of nerve fibroblasts is Thy1
(Assouline et al., 1983
;
Brockes et al., 1979
;
Morris and Beech, 1984
;
Vroemen and Weidner, 2003
). We
found that although 46±17% (mean±s.d.) of the cells with
myofibroblast morphology that arose within nerve NCSC colonies in 14-day
standard cultures were SMA+Thy1, a similar
proportion (44±15%) were SMA+Thy1+, and
10±3% were SMAThy1+
(Fig. 8A). Thus a high
proportion of SMA+ myofibroblasts also expressed Thy1, and some
myofibroblasts acquire a SMAThy1+ phenotype
similar to endoneurial fibroblasts in vivo
(Morris and Beech, 1984
).
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To test whether endoneurial fibroblasts in postnatal mouse nerves also
formed SMA+ myofibroblast colonies in culture, we sorted and
cultured Thy1+ and Thy1 cells from the sciatic
nerves of postnatal Wnt1-Cre+loxpRosa+ mice. Most of the
cells that formed ß-gal-expressing myofibroblast-only colonies in culture
came from the Thy1+ cell fraction, and virtually all of the cells
in such colonies expressed SMA after 5 to 7 days of culture (not shown).
Interestingly, expression of SMA in culture was again associated with
downregulation of Thy1 expression. As the only neural crest-derived
fibroblasts in the nerve are endoneurial fibroblasts, these data suggest that
Thy1+ endoneurial fibroblasts downregulate Thy1 expression and form
SMA+ myofibroblast colonies in culture. Consistent with this, a
variety of fibroblasts have been observed to adopt a SMA+
myofibroblast phenotype in culture and after injury in vivo
(Sappino et al., 1990).
Together these results suggest that the neural crest-derived myofibroblasts
that arise in culture correspond to cells that acquire an endoneurial
fibroblast fate in vivo.
The regulation of fibroblast and Schwann cell differentiation from NCSCs
To begin to study the mechanism by which the nerve environment regulates
fibroblast and Schwann cell differentiation from nerve NCSCs, we investigated
whether known factors from the nerve environment could account for this
multilineage differentiation. First, the factors would have to cause
individual sciatic nerve NCSCs to form Schwann cells and myofibroblasts, but
not neurons, as sciatic nerve NCSCs do not give rise to neurons in the nerve
(Bixby et al., 2002). Second,
these factors would have to be permissive for neurogenesis from gut NCSCs, as
gut NCSCs consistently form neurons when transplanted into developing
peripheral nerves, and fail to form glia in most cases
(Bixby et al., 2002
).
Neuregulin (Nrg1) (Dong et al.,
1995
), Delta, and Bmp4 are known to be expressed in developing
peripheral nerves (Bixby et al.,
2002
), so we examined how this combination of factors affected the
differentiation of NCSCs.
In standard medium (No Add; Table
3), 73% of sciatic nerve NCSC colonies were multilineage
(Table 3). Addition of the
immunoglobulin Fc domain protein [a negative control for the addition of
Delta-Fc (Morrison et al.,
2000b)] did not affect differentiation when compared with standard
medium. Bmp4 by itself significantly promoted neurogenesis, whereas Delta-Fc
and GGF by themselves significantly promoted gliogenesis
(Bixby et al., 2002
;
Morrison et al., 2000b
;
Morrison et al., 1999
;
Shah et al., 1996
;
Shah et al., 1994
). Note that
plating efficiencies (the percentage of cells that survived to form colonies)
varied among treatments because Nrg1 promotes the survival of nerve NCSCs
(Morrison et al., 1999
). Most
colonies contained neurons in the combination of Bmp4 with Nrg1
(Shah and Anderson, 1997
). The
combination of Bmp4 and Delta-Fc promoted glial and myofibroblast
differentiation by nerve NCSCs, as had been previously observed
(Morrison et al., 2000b
).
Despite the fact that each individual factor promoted only neurogenesis or
gliogenesis, the combination of Bmp4, Nrg1 and Delta-Fc generated the highest
proportion of colonies (52%) that contained both myofibroblasts and glia (M+G;
Table 3; see Fig. S6 in
supplementary material). Almost all remaining colonies in this treatment
contained only glia (G-only) or only myofibroblasts (M-only). This
demonstrates that the combination of Bmp4, Nrg1 and Delta-Fc caused sciatic
nerve NCSCs to differentiate into glia and myofibroblasts (see Fig. S6),
consistent with the possibility that these factors in the nerve environment
promote the differentiation of Schwann cells and endoneurial fibroblasts from
NCSCs.
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To test whether these factors caused sciatic nerve NCSCs to undergo lineage restriction, we cultured nerve NCSCs (isolated by flow-cytometry) at clonal density either in standard medium, or in medium supplemented with Bmp4, Nrg1 and Delta-Fc, for 7 days, and then subcloned the colonies into secondary cultures that contained standard medium. After 14 days, we examined the composition of the secondary colonies. Of 22 colonies that were subcloned from standard medium, 19 gave rise to multilineage secondary colonies, averaging 63±73 multilineage secondary colonies per primary colony, in addition to various other types of secondary colonies. This indicates that in standard medium NCSCs give rise to colonies that retain substantial numbers of multipotent progenitors. By contrast, colonies cultured in medium supplemented with Bmp4, Nrg1 and Delta-Fc for 7 days never gave rise to multilineage secondary colonies or any secondary colonies that contained neurons. Rather all 35 colonies subcloned from this treatment gave rise to M+G, G-only, and/or M-only colonies. This indicates that the combination of Bmp4, Nrg1 and Delta-Fc caused nerve NCSCs to undergo lineage restriction in culture to form progenitors that lacked neuronal potential, but retained glial and/or myofibroblast potential.
Bmp4+Nrg1+Delta-Fc promote the acquisition of an endoneurial fibroblast phenotype
If these factors promote the differentiation of NCSCs into endoneurial
fibroblasts in vivo, then they would be expected to increase the proportion of
myofibroblasts that acquire a SMAThy1+ phenotype
in culture. To test this, we cultured NCSCs under standard conditions or in
the presence of standard medium supplemented by Bmp4, Nrg1 and Delta-Fc for 6
days, and then stained with antibodies against Thy1 and SMA. Under standard
conditions, only 37% of colonies contained myofibroblasts after 6 days
(averaging 5.9/colony) and 92±6% of cells with myofibroblast morphology
were SMA+Thy1, indicating poor differentiation
toward an endoneurial fibroblast phenotype
(Fig. 8B,D). However, after 6
days in Bmp4, Nrg1 and Delta-Fc, 94% of colonies in these experiments
contained myofibroblasts (as well as glia in most cases). and the number of
myofibroblasts per colony was greatly increased (averaging 1073/colony).
Almost 90% of cells with a myofibroblast morphology expressed Thy1 under these
conditions. Of these, approximately one-third were SMA, and
two-thirds were SMA+ (Fig.
8C,D). All of these values were significantly different from what
was observed in standard medium (P<0.05). These data suggest that,
in addition to increasing the proportion of NCSCs that form myofibroblasts and
glia in culture, the combination of Bmp4, Nrg1 and Delta-Fc also dramatically
increases the number of myofibroblasts that express Thy1.
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Discussion |
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The fact that similar percentages of Schwann cells and endoneurial fibroblasts expressed ß-gal, whether the fate mapping was performed using Wnt1-Cre (Table 1) or Dhh-Cre (Fig. 5), also supports the conclusion that these cell populations originated from a common progenitor within the nerve. Wnt1 is expressed at the onset of neural crest migration, whereas Dhh is not expressed until days later in developing peripheral nerves. If Schwann cells and endoneurial fibroblasts arose from independent lineages of progenitors, these independent lineages would have to simultaneously begin expressing Wnt1, and then later simultaneously express Dhh. Moreover, the levels of expression of these genes would also have to be similar in both lineages to account for the similar degrees of recombination in both cell types. A more parsimonious explanation is that these lineages arise from a common progenitor that does not undergo lineage commitment until after migrating into the developing nerve.
Implications for models of nerve development
The finding that NCSCs undergo multilineage differentiation in developing
peripheral nerves indicates that nerve development is more complex than was
previously thought. Prior models of neural crest differentiation in the nerve
considered only the overt differentiation of Schwann cells from Schwann cell
precursors (Jessen et al.,
1994; Jessen and Mirsky,
1992
; Mirsky and Jessen,
1996
). In the context of such models, genes that regulate neural
development were assumed to play a role in this differentiation process. We
find that NCSCs self-renew within developing peripheral nerves
(Morrison et al., 1999
),
undergo restriction to form non-neuronal progenitors, make a fate choice
between the glial and myofibroblast lineages, and then differentiate into
Schwann cells and endoneurial fibroblasts
(Fig. 9). The existence of
these partially restricted, and glial and myofibroblast committed progenitors
in vivo is supported by the ability to culture glia+myofibroblast (G+M)
colonies, glia-only (G-only) colonies, or myofibroblast-only (M-only) colonies
directly from freshly dissociated sciatic nerve
(Morrison et al., 1999
).
Progenitors that form each of these colony types also arise from individual
sciatic nerve NCSCs upon subcloning in culture
(Morrison et al., 1999
). It
will be necessary to consider the functions of genes that are expressed by
neural progenitors in the nerve in terms of NCSC self-renewal, lineage
restriction, lineage commitment, and differentiation.
These results are also of general importance to understanding neural
development, as they demonstrate that trunk neural crest cells actually adopt
a myofibroblast fate in vivo. Previously, neural crest cells were known to
form smooth muscle in the cardiac outflow tracts but more caudal trunk neural
crest cells were not thought to adopt similar fates. As a result, although
trunk NCSCs, including sciatic nerve NCSCs, have been defined partly based on
their ability to form myofibroblasts (in addition to neurons and glia) in
culture (Bixby et al., 2002;
Morrison et al., 1999
;
Shah et al., 1996
), it was
unclear whether this myofibroblast capacity was actually used in vivo. This
question has gained added importance with the demonstration in the developing
spinal cord that separate populations of progenitors form oligodendrocytes and
motoneurons, as compared to astrocytes and interneurons
(Gabay et al., 2003
). This
suggests that although stem cells from the spinal cord have been defined based
on their ability to form neurons, astrocytes and oligodendrocytes, there may
not be a single cell population in vivo that actually forms all of these cell
types in the spinal cord. The finding that the trunk neural crest gives rise
to endoneurial fibroblasts (Fig.
4), in addition to neurons and glia (see Fig. S2 in supplementary
material), demonstrates that the ability of these cells to form myofibroblasts
does not result from reprogramming in culture. It is thus of interest to study
the fibroblast/glial fate decision in addition to studying the neuron/glia
fate decision by NCSCs.
The mechanism by which nerve NCSCs undergo multilineage differentiation
appears to involve the combinatorial action of Bmp4, Delta,and Nrg1 on the
NCSCs. Each of these factors is expressed in developing peripheral nerves in
vivo (Bixby et al., 2002;
Dong et al., 1995
). Together
these factors cause sciatic nerve NCSCs to form glia and myofibroblasts, but
not neurons, in culture (Table
3, see Fig. S6 in supplementary material), even though
individually the factors promote neuronal or glial fate determination
(Morrison et al., 2000b
;
Morrison et al., 1999
;
Shah et al., 1996
;
Shah et al., 1994
). In
cultures supplemented with Bmp4, Delta and Nrg1, sciatic nerve NCSCs formed
glia+myofibroblast (G+M) colonies, glia-only (G-only) colonies, and
myofibroblast-only (M-only) colonies (Table
3), consistent with the possibility that these factors regulate
the differentiation of NCSCs in developing nerves. Moreover, in the presence
of these factors, NCSCs generated much larger numbers of myofibroblasts, and
the myofibroblasts were more likely to acquire a
Thy1+SMA phenotype, thus resembling endoneurial
fibroblasts (Fig. 8). The
involvement of these factors is further supported by the observation that they
cause gut NCSCs to give rise to neurons and myofibroblasts, but usually not
glia, in culture (Table 3),
consistent with the observation that gut NCSCs give rise to neurons but
usually not to glia upon transplantation into developing peripheral nerves
(Bixby et al., 2002
). Thus the
combinatorial effects of Bmp4, Delta and Nrg1 on sciatic nerve and gut NCSCs
in culture appear to be consistent with the way in which these NCSC
populations differentiate in developing nerves in vivo.
The embryonic origin of nerve cell types
The embryonic origin of different nerve cell types has long been
controversial but until recent years it was not possible to directly trace the
origin of nerve cell types in fate-mapping experiments in vivo. For example,
it was debated whether perineurial cells were derived from Schwann cells,
endoneurial fibroblasts, or an independent lineage of fibroblasts, based on
morphological criteria (Low,
1976; Peltonen et al.,
1987
; Peters et al.,
1976
). Our fate mapping experiments demonstrate directly that the
vast majority of perineurial cells are not neural crest derived and therefore
cannot be lineally related to Schwann cells or endoneurial fibroblasts. Our
data leave open the possibility that a small minority of perineurial cells on
the endoneurial surface of the perineurium could be neural crest derived. The
fibroblasts that give rise to the perineurium
(Bunge et al., 1989
) may be
mesodermally derived, but our data do not address this directly. As nerve
pericytes are also not neural crest derived they must invade the endoneurial
space along with blood vessels. Whether pericytes are mesodermally-derived or
whether they are lineally related to perineurial cells is not addressed by our
data. Because Schwann cells secrete signals that regulate the formation of
perineurium (Parmantier et al.,
1999
), nerve development involves a complex interaction of neural
crest-derived and non-neural crest-derived progenitors.
Implications for nerve pathologies
Our finding that endoneurial fibroblasts are neural crest derived may also
help to understand nerve pathology. Neurofibromas and malignant peripheral
nerve sheath tumors often contain fibroblasts in addition to cells that
resemble Schwann cells (Serra et al.,
2000; Takeuchi and Uchigome,
2001
). Some of these fibroblasts derive from surrounding normal
tissue rather than arising from the neoplastic clone
(Serra et al., 2000
). However,
neoplastic transformation of NCSCs or non-neuronal restricted nerve
progenitors could potentially yield clonal tumors containing both Schwann
cells and fibroblasts. Recently, neurofibromas were concluded to arise from
Schwann cells, based on their ability to generate neurofibromas following
conditional deletion of floxed NF1 using Krox20-Cre
(Zhu et al., 2002
). However,
Krox20 also appears to be expressed by sciatic nerve NCSCs (data not shown),
so this conditional deletion strategy would be expected to yield NF1-deficient
NCSCs as well as Schwann cells. Our observation that endoneurial fibroblasts
are derived from nerve NCSCs raises the possibility that NCSCs are sometimes
transformed by NF1 deficiency to form tumors containing clonally-derived
Schwann cells and fibroblasts.
Neural differentiation in the nerve thus involves stem cell self-renewal, lineage restriction, lineage commitment and multilineage differentiation. We have begun to elucidate this process by showing that the combination of Bmp4, Delta and Nrg1 promotes glial and fibroblast differentiation by sciatic nerve NCSCs. This changes models of nerve development by broadening the scope of neural crest involvement.
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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/131/22/5599/DC1
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