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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02852
1 Max Planck Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
2 Department of Anatomy, TU Dresden, Fetscherstrasse 74, D-01307 Dresden,
Germany.
* Author for correspondence (e-mail: tanaka{at}mpi-cbg.de)
Accepted 15 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Axolotl, Spinal cord, Regeneration, Neural progenitors, PAX6, PAX7, Single-cell labeling, Tissue grafting
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; O'Hara et al.,
1992). Upon amputation and lesioning of the spinal cord, the
terminal radial glial cells polarize toward the tail tip and apparently
undergo an epithelial to mesenchymal transition to migrate toward the cut
surface (O'Hara et al., 1992).
These cells then form a single-cell-layered tube of neuroepithelial cells
called the ependymal tube that extends posteriorly in concert with overall
tail regeneration. After a significant period of growth, cellular
differentiation occurs in a rostral to caudal sequence, with the tip of the
tail remaining undifferentiated until later stages of regeneration
(Arsanto et al., 1992).
Cell tracking experiments confirm that the radial glial cells are the
progenitor cells for spinal cord regeneration. Electroporation into luminal
cells of a plasmid where enhanced green fluorescent protein (eGFP) expression
was driven by the glial fibrillary acidic protein (GFAP) promoter resulted in
transfection of radial glial cells. When these cells were monitored in live
animals over time, they contributed to the regenerating ependymal tube,
underwent some proliferation and generated, among other cell types, neurons
(Echeverri and Tanaka, 2002).
In a separate set of studies, the contribution of differentiated neurons to
the regenerating spinal cord was tested by retrogradely labeling motor neurons
with rhodamine dextran prior to tail amputation
(Zhang et al., 2003). Labeled
neurons were swept along with the growing ependymal tube, and came to reside
in the regenerate. There was no evidence of dedifferentiation of these neurons
and so this cell source could not account for the growth and formation of all
the new neurons in the regenerate.
Although the basic cell tracking experiments indicated that radial glial cells are involved in regeneration, these studies did not address how the progenitor cells produce the various neuronal cell types during regeneration. Several scenarios are possible. First, the spinal cord may harbor highly multipotent stem cells that undergo expansion and populate all regions of the regenerating spinal cord to form all the different cell types. This situation would necessitate the presence of inducing factors that direct the cells to form the different cell types. Second, the spinal cord may contain highly restricted progenitors that are committed to forming specific neuronal cell types. Third, multipotent stem cells may, under normal regeneration conditions, tend to divide within a local domain, and thus could contribute to a limited spectrum of cell types even though they have the potential to form other cell types if induced by strong extracellular cues. Fourth, regeneration may depend on a mixture of multipotent and restricted progenitor cells.
The different neuronal subtypes such as sensory, inter-, commissural and
motor neurons derive from distinct domains along the dorsal, lateral and
ventral axis of the developing spinal cord. These domains are molecularly
defined by a combinatorial code of transcription factors expressed in the
progenitor cells well before neuronal differentiation (for reviews, see
Briscoe and Ericson, 2001;
Shirasaki and Pfaff, 2002;
Tanabe and Jessell, 1996).
During embryogenesis signals from outside the neural tube direct uncommitted
neural progenitor cells to form these domains. Namely, notochord-derived sonic
hedgehog (SHH) and ectodermally derived bone morphogenetic proteins (BMPs) are
crucial extracellular signaling molecules for these patterning events.
By contrast, grafting experiments in the regenerating axolotl tail indicate
that the information for establishing the dorsal/ventral (D/V) domains of the
regenerating spinal cord comes from within the mature spinal cord. Holtzer
removed a section of spinal cord from the mature tail and then regrafted it
after rotating it 180° in the D/V axis
(Holtzer, 1956). Upon healing,
the tail was cut through the graft to induce regeneration. Under these
conditions, the entire tail regenerate including neural, cartilage and muscle
structures was inverted 180° along the D/V axis. This experiment
indicated, first, that the information for organizing the D/V patterning of
the regenerating spinal cord derives from the mature spinal cord. Second, it
indicated that factors from the spinal cord determine the patterning of the
surrounding tissues. Schnapp et al. defined some of the molecular factors
underlying these properties (Schnapp et
al., 2005). They showed that the transcription factors MSX1, PAX7
and PAX6, which delineate dorsal, dorso-lateral and lateral progenitor cell
domains in the developing neural tube, were expressed in the uninjured axolotl
spinal cord. This indicates that the mature spinal cord contains molecularly
defined progenitor cell domains along the D/V axis similar to those found in
development. These domains were also found in the regenerating ependymal tube.
The secreted signaling factor sonic hedgehog was expressed in the ventral-most
floorplate spinal cord cells both in the mature and regenerating tissue. The
activity of the sonic hedgehog was shown to be required for determining the
PAX7 domain size in the spinal cord, the induction of cartilage ventral to the
regenerating spinal cord, and for tail blastema cell proliferation. Although
the Schnapp et al. work defined the molecular players underlying progenitor
cell identity during spinal cord regeneration, there are still many unanswered
questions concerning how the progenitor cells come to regenerate the correct
complement of cell types in the spinal cord. A prime missing aspect is how the
progenitor cells populate the regenerating spinal cord, and whether a single
progenitor cell will produce descendents that will populate all D/V regions of
the spinal cord, or whether cells remain within their original domain. It is
also not known on a single cell level how cells proliferate to allow posterior
extension of the regenerating ependymal tube.
Here we examine these issues by tracking cells both at the clonal cell level and in cell groups. We first determine the size of the mature spinal cord region that gives rise to the regenerating spinal cord. Second, we examine the spatial distribution of clone growth along the anterior/posterior (A/P) and D/V axis. We find that although most cells grow along the A/P axis and remain close to their D or V origin, some cell clones expand to produce progeny that populate multiple D/V areas. These cells can change their transcription factor expression. Interestingly, we found that the posterior-most region of the regenerating spinal cord, termed the terminal vesicle, is molecularly and cellularly distinct from the other regions. The cells there are PAX7- and PAX6-. Also, in this region, ventrally located cells spread into the dorsal region and exit the spinal cord into the blastema.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). After 7 days of healing, tails were amputated and
imaged weekly on an Olympus SZX 12 stereomicroscope using a Spot imaging
system (Diagnostic Imaging Systems).
Embryonic transplantations and lineage tracing
Orthotopic transplantations of prospective floor plate or roof plate
stripes between eGFP transgenic axolotl donors (d/d) and white (d/d) hosts
(both stage 15-16) were performed (Fig.
8) in 1x Steinberg's solution
(Steinberg, 1957) supplemented
with antibiotics (penicillin/streptavidin/amphotericine). Embryos were grown
to 2 cm larvae at room temperature (RT). Tails were then amputated and
photographed every 2-3 days using the imaging protocols described below.
Electroporation
Cells were electroporated by cutting the tail of 2 cm-long larval axolotls
and inserting a DNA-filled electrode into the spinal cord lumen, as described
by Echeverri and Tanaka (Echeverri and Tanaka, 2003). To transfect the dorsal
cells in the axolotl tail spinal cord, the animals were orientated with the
dorsal side to the ground electrode. To transfect DNA into single cells,
optimum electroporation conditions were three pulses (50 V, 200 Hz and a pulse
length of 100 mseconds), applied using an SD9 Stimulator (Grass Telefactor,
West Warwick, RI).
Analysis of lineage tracing experiments
In these lineage tracing experiments, a total of approximately 2000
axolotls were electroporated with cytoplasmic eGFP, nuclear DsRed (data not
shown) or nuclear GFP plasmids. From approximately 800 axolotl larvae
electroporated with nuclear GFP, 77 had clearly identifiable single cells and
were used for experimental observation. In 55 (60%) of these cases cells
disappeared or did not divide during the course of observation. In 22 (40%)
animals it was possible to follow dividing cells during regeneration.
Imaging of labeled cells
Axolotl larvae containing labeled cells were imaged every 2-3 days by
anesthetizing the animals in 0.01% Ethyl p-aminobenzoate and placing them on a
coverslip. eGFP-expressing cells were imaged using a Zeiss Axiovert 2
microscope controlled by a Metamorph image acquisition system. Cells were
imaged using Zeiss Water Ph2 Plan-Neofluar 25x/0.80 Imm Korr and
40x/1.2 W Korr objectives, and the tails were taped to the coverslip to
minimize the distance between the cells and the objective.
Fixation and cryosectioning
For PAX7 and PAX6 immunostaining, larval axolotl tail samples were fixed in
freshly made 4% paraformaldehyde (PFA) at RT, then overnight (ON) at 4°C.
They were then washed 2x30 minutes in 1xPBS and placed in a
solution of 30% sucrose/1xPBS ON at 4°C. For sectioning, the samples
were embedded in TissueTek (Sakura).
For adult tail and body spinal cord immunostaining, adult axolotls were perfused through the heart with 1xPBS followed by freshly made 4% PFA. Spinal cord pieces were dissected free from surrounding tissue and placed in 10% and 20% sucrose/1xPBS ON at 4°C, and then into 20% sucrose/3.5% gelatin (Bloom 80-120; Merck)/1xPBS ON at 37°C and embedded in 20% sucrose/7.5% gelatin/1xPBS. Cryosections of TissueTek- and gelatin-embedded samples were collected on Histobond Adhesion microslides (Marienfeld). The sections were allowed to air dry for 2-3 hours and were immunostained as described below.
Immunolabeling
Monoclonal antibodies against axolotl PAX6 were generated by producing a
GST-fusion protein against the axolotl PAX6 protein corresponding to amino
acid region 70-421 of the mouse protein. The protein was injected into mice
(EMBL facility) and resulting hybridoma clones were screened initially by
ELISA, and then on cryosections. PAX6 IgG was purified from hybridoma cell
supernatant by binding to a HiTrap Protein G column (GE Healthcare) and
elution at pH 2. Purified anti-PAX6 IgG was conjugated with
N-hydroxysuccinimidyl ester-digoxygenin (NHS-DIG) in 0.1 M carbonate
buffer, pH 9, at a labeling stoichiometry of 20:1 (DIG:AB). After labeling
reactions, PAX6-DIG was dialyzed overnight at 4°C in 1xPBS using
Spectra/Pore membrane MWCO 6000-8000.
For PAX7/PAX6 double immunolabeling, the cryostat sections were incubated ON at 4°C in block buffer (1xTBS, 0.03% Triton X-100, 10% rabbit or goat serum), incubated with primary PAX7 antibody (Developmental Studies Hybridoma Bank), washed with wash buffer (1xTBS, 0.03% Triton X-100), incubated with secondary antibody Cy5 Fab fragments (goat anti-mouse; Dianova), washed well with wash buffer, incubated with PAX6-DIG antibody, washed with wash buffer, incubated with sheep anti-DIG-rhodamine Fab fragments (11 207 750 910; Roche) and stained with 1 µg/ml Hoechst. All primary and secondary antibodies were diluted in block buffer and each antibody reaction was done ON at 4°C.
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All animal procedures were performed in adherence to the guidelines regulating work at the MPI-CBG.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (Fig. 1A,D).
After 7 days of healing, the tails were amputated at different distances
ranging from 300 to 3500 µm from the rostral junction between the host and
donor spinal cord (Fig. 1B,E,
Table 1). The length of
remaining eGFP-positive spinal cord was measured at 2 days post-amputation to
account for tissue loss because of the injury
(Fig. 1B,E,
Table 1). At day 9 and day 16,
the total length of the eGFP-expressing region versus the total length of the
regenerating spinal cord was measured (Fig.
1C,F, Table 1). The
amputation plane could be determined as the point where the segmented
cartilage changes to the newly forming cartilage rod
(Fig. 1, broken line).
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Although this experiment defined the source of regenerating neural progenitors and the uniform proliferation along the spinal cord, it did not give insight on how clones deriving from individual progenitor cells expand during tail regeneration. Considering the posterior growth of the regenerating spinal cord, we expected significant extension of clones along the A/P axis. More importantly, we wanted to understand whether a single progenitor cell may produce descendents that form diverse neural cell types. Because neural cell types form in characteristic positions along the D/V axis of the spinal cord, we endeavored to label a cell in a defined dorsal or ventral position, follow its division and the position of its descendents and then finally to determine whether the daughters expressed different molecular markers reflecting different identities.
Schnapp et al. had demonstrated the existence of a dorsal PAX7+,
lateral PAX6+ and a ventral SHH+ domain in both the
uncut and the regenerating spinal cord, providing the essential markers
required for our study (Schnapp et al.,
2005). Prior to undertaking lineage analysis, we extended the
expression analysis of PAX7 and PAX6 to examine the variation of the
expression along the A/P axis of the regenerating spinal cord.
PAX7 and PAX6 mark dorsal and lateral domains in the axolotl spinal cord but are absent from the terminal vesicle
In the previous study PAX6 was examined via in situ hybridization
(Schnapp et al., 2005). In
order to ultimately combine our lineage tracing studies described below with
PAX6 localization, we generated a monoclonal antibody against the axolotl PAX6
protein that we use for the current localization studies.
To determine the A/P dependence of the PAX7 and PAX6 expression, we examined immunolabeled cross-sections at different A/P levels of the regenerating spinal cord spanning the terminal vesicle to the amputation plane in 2 cm-long larvae. At the very caudal terminus of the regenerating spinal cord, PAX7 was barely detectable (Fig. 2A,C, arrowhead), but expression was found in an increasing number of cells toward the amputation plane (Fig. 2D,F,G,I). Mature spinal cord showed strong PAX7 expression relative to regenerated spinal cord regions (Fig. 2J,L). PAX6 expression was also not detectable close to the terminal vesicle (Fig. 2A,B) but was expressed in the lateral domains of the regenerating tail spinal cord in more rostral regions (Fig. 2D,E). Closer to the amputation plane, PAX6 and PAX7 were co-expressed in some dorso-lateral cells (Fig. 2G,H, yellow arrows). In mature tissue PAX6 was expressed in lateral and all dorsal domain cells, so that in the dorsal region, all PAX7+ cells were PAX6+ (Fig. 2J,K). These expression patterns were observed at every timepoint of regeneration. The strong PAX7 and PAX6 expression in mature tail spinal cord suggests that progenitor cells with D/V identity reside in the mature tissue.
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) was performed on 2 cm-long larvae, which represents the
stage where our experimental lineage analysis was carried out. We were
concerned whether the expression in the mature region represented a situation
specific to an animal that was still undergoing rapid growth. We therefore
examined PAX7 and PAX6 distribution in the spinal cord of fully grown adults.
We found PAX7+ and PAX6+ cells in the spinal cord of the
adult body (Fig. 3A) and
uninjured tail (Fig. 3D) spinal
cord. Interestingly, in the adult body spinal cord the PAX7+ cells
were found in a layer of cells (Fig.
3A, arrowheads) that was clearly distinct from the ependyma and
the neuronal layer as defined by ßIII-tubulin immunostaining
(Fig. 3B) and Neuronal Nuclei
(NeuN) staining (Fig. 3C). We
did not observe any clear co-expression of PAX7 and ßIII-tubulin (an
early neuronal marker), suggesting that the PAX7-positive cells probably
represent a progenitor cell in the mature spinal cord. PAX6 expression was
restricted to the lateral cells of the mature body and tail spinal cord
ependymal layer. These results indicate that spatially restricted PAX7 and
PAX6 expression is a feature of the fully adult spinal cord.
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Fig. 4 shows a ventral spinal cord cell that produced a clone spanning ventral, lateral and dorsolateral domains. On day 5 after amputation and electroporation the cell divided (Fig. 4B) and continued to divide (Fig. 4D-F) so that by day 23 (Fig. 4F) the cell had generated a 12-cell clone that occupied ventral, lateral and dorso-lateral regions of the regenerated spinal cord. On day 23 this sample was fixed for cryosectioning and PAX7/PAX6 double immunostaining to analyze the marker profile of the daughter cells (Fig. 5). The clone consisted of both PAX6- cells (Fig. 5A,B, arrows) and PAX6+ cells (Fig. 5D,E, arrows with asterisks), indicating that a single cell had produced daughters with different identities.
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The behavior of the 21 clones is summarized in Table 2 and the images of the clones are included in supplementary material Figs S1-S19. In Table 2, the grey bars span the days on which the clone was followed either before the cells lost expression or the clone was fixed for analysis. The numbers within the bars denote the number of cells in the clones on a given day. From this data we find that the average clone size was 4, with a range of 2 to 12 cells. Because a cell within a clone would sometimes disappear during observation, this number represents the maximum number of cells in the given clone during its observation period. Disappearance of cells could either be because of dilution of plasmid and loss of nucGFP expression or cell death. The average time of observation of the clones was 17 days. The fourfold expansion of the clones is roughly consistent with the overall growth of the spinal cord when we consider that we initially amputated approximately 2-3 mm from the spinal cord and considering our evidence that the regenerated portion of the spinal cord derives from a 500 µm length of the spinal cord.
From these single-cell lineage tracing experiments we conclude that most clones remain associated with their original D/V domains but that some progenitors can give rise to descendents that switch domains. These descendents can express distinct D/V markers (Fig. 7), indicating the action of inductive signals in the regenerating axolotl spinal cord that regulate D/V patterning during regeneration. We observed no defect in the animals harboring multipotent clones that might indicate domain switching was induced by experimental circumstances.
Lineage tracing experiments using cytoplasmic-eGFP showed results that were
very similar to the nuclear-eGFP experiments
(Table 3). However, the
cytoplasmic-eGFP experiments were not grouped together with the nuclear-eGFP
results because of the technical limitations of unambiguously identifying a
single cell in vivo at the beginning of lineage tracing. It was, however,
possible to follow the division of a cell (or cells) that began in a specific
region and the distribution of their descendents (supplementary material Fig.
S19). From 18 animals used for this analysis, seven (
40%) of the cell
groups remained in one region, whereas in 11 (60%) the cells changed
between the different domains (Table
3).
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Fig. 9 illustrates a sample with dorsal cell labeling, whereas Fig. 10 depicts a ventrally labeled animal. The tails were amputated close to dorsally or ventrally restricted eGFP+ cell populations (Fig. 9A, Fig. 10A). Dorsal labeling showed that dorsal/dorso-lateral cell groups proliferated and expanded within dorsal and dorso-lateral domains in the regenerating spinal cord (Fig. 9A-C).
Tracing of ventral/ventro-lateral cell populations mainly showed spatial restriction of cells to ventral and ventro-lateral domains during regeneration (Fig. 10A-C). Strikingly, in several ventral cell tracking experiments (3/5) the cells lying close to the terminal vesicle eventually distributed to the dorsal domain during the time of observation (Fig. 10C,E, arrowheads). These new dorsal cells seemed to migrate out of the terminal vesicle (Fig. 10D, arrowheads). Thus, through these embryonic transplantation experiments we conclude that dorsal and ventral cells generally remain distinct, except when close to the terminal vesicle where cells can relocate from a ventral to a dorsal domain.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Our
results indicate that at least some of the neural progenitors in the
regenerating spinal cord are multipotent, but they do not resolve whether all
progenitor cells are multipotent or if the regenerating spinal cord consists
of a mixture of multipotent and lineage-restricted progenitor cells. Precise
D/V transplantation experiments to test this issue are technically
demanding.
In vivo observations pointing toward a multipotent stem cell
It is interesting to consider our results in the light of those from Gabay
et al., who have analyzed D/V identity in cultured neural stem cells in vitro
(Gabay et al., 2003). They
noted that in vivo, oligodendrocyte precursors derive from ventral neural tube
regions whereas astrocyte precursors derive from a distinct dorsal domain,
implying that individual neural progenitor cells must generate at most two
cell lineage types in vivo. By contrast, trilineage multipotent stem cells are
observed in in vitro cultures from the same tissue. They investigated this
discrepency and showed that dorsally derived progenitors were ventralized by
fibroblast growth factor (FGF) and sonic hedgehog when cultured in vitro, thus
leading to tripotency. The authors question the relevance of trilineage stem
cells that derive from such conversions in vivo. Our cell tracing experiments
show that in vivo, during outgrowth of the ependymal tube, cells from a dorsal
or ventral domain can switch to an alternate location and acquire a new
identity, as assayed by PAX6 and PAX7 in these studies. Although we have not
demonstrated whether our progenitor cells generate the three lineages
(oligodendrocyte, astrocyte and neuron), our results strongly indicate that
the kind of conversion between dorsal and ventral identities observed by Gaby
et al. in culture are relevant to in vivo stem cell behaviors.
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Contribution of spinal cord cells to tissues outside of the spinal cord
A fascinating aspect of terminal vesicle cell identity is the acquisition
of migratory properties. Through our embryonic grafting experiments, we
clearly observed a trail of eGFP+ cells leaving the dorsal surface
of the terminal vesicle into the surrounding blastema during tail
regeneration. This phenomenon accounts for the disappearance of
eGFP+ terminal vesicle cells in our spinal cord transplantation
experiments. When we produced a chimeric regenerating spinal cord with an eGFP
cell label only in the distal-most region of the regenerating spinal cord, the
eGFP label had disappeared after 16 days. This is presumably because of the
migration of all the labeled cells into the blastema. Indeed, in these spinal
cord transplants and in the embryonic tissue-grafted animals, we see abundant
eGFP+ cells outside the spinal cord, including blood vessels,
apparently in Schwann cells and occasionally in muscle and cartilage. However,
both of these grafts are not pure enough to allow us to conclude which of
these cell types derive from bonafide neural progenitor cells.
The terminal vesicle has in fact long been characterized as a region where
an epithelial to mesenchymal transition occurs, and where cells are proposed
to delaminate from the ependymal tube to join the surrounding blastema tissue
(Benraiss et al., 1997;
Egar and Singer, 1972;
O'Hara et al., 1992). Our
eGFP+ spinal cord transplantation and the eGFP+
embryonic neural tube grafting experiments allowed us to confirm that cells
escape the regenerating spinal cord from this region, as we observe trails of
cells emanating from the posterior-dorsal wall of the terminal vesicle.
Clearly an important question is what these migrating cells form during
regeneration. The epithelial to mesenchymal transition is reminiscent of
embryonic neural crest. Benraiss et al., using biolistic transfection of an
alkaline phosphatase expression vector into the newt spinal cord, found
alkaline phosphatase-expressing cells emanating from the ependymal tube
(Benraiss et al., 1997). At
later timepoints alkaline phosphatase expression was observed in melanophores
and Schwann cells. We are currently working to confirm whether the primary
fate of the migrating terminal vesicle cells is neural crest. Because the cell
tracking experiments were performed in white mutant animals (d/d alleles),
which are deficient in melanophore migration and survival, it was not possible
to assay the contribution to melanophores in the current experiments.
A further question is whether the migrating cells contribute to cell types
outside of classical neural crest lineages. Echeverri and Tanaka reported the
formation of muscle and cartilage after electroporation into the spinal cord
of an expression plasmid where eGFP was driven by the GFAP promoter
(Echeverri and Tanaka, 2002).
Considering the frequency of the events reported in this study, we expected a
higher number of muscle fibers to be labeled after our spinal cord
transplantation experiments. The basis for the discrepency between the two
experimental results is not clear. One possibility is that the electroporation
technique itself induced an increase in cellular plasticity or a tendency to
fuse with other cell types. The injury caused by insertion of the glass
microcapillary could have caused such events. Another possibility is the use
of transient transfection in the electroporation experiments versus the cells
from the transgenic animals. One issue is whether the integrated transgene can
become silenced during the process of cellular plasticity. For example, such
silencing phenomena were observed in cells from a transgenic mouse where eGFP
was driven by the same ubiquitous CAGGs promoter
(Torensma and Figdor, 2004).
eGFP was robustly expressed in mature lymphocytes but was barely expressed in
immature thymocytes. We are currently investigating these issues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2083/DC1
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ACKNOWLEDGMENTS |
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