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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02573
1 Zebrafish Neurogenetics Junior Research Group, Institute of Virology,
Technical University-Munich, Trogerstrasse 4b, D-81675, Munich, Germany and
GSF-National Research Center for Environment and Health, Department Zebrafish
Neurogenetics, Institute of Developmental Genetics, Ingolstaedter Landstrasse
1, D-85764 Neuherberg, Germany.
2 Developmental Biology Unit, Department of Biology I, University of Freiburg,
Hauptstrasse 1, D-79104 Freiburg, Germany.
* Authors for correspondence (e-mail: chapouton{at}gsf.de; bally{at}gsf.de)
Accepted 8 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neural stem cell, Zebrafish, her5, E(Spl), Midbrain-hindbrain boundary
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Understanding the mechanisms underlying the survival and the regenerative
properties of neural stem cells would, however, be of tremendous value.
To date, our knowledge on adult neurogenesis is largely derived from
analyses of the vertebrate forebrain. In birds and reptiles, proliferation
takes place continuously over the lifetime within the telencephalon, and newly
arising neurons survive and are integrated into functional circuits seasonally
(Garcia-Verdugo et al., 2002).
In rodents, many studies have concentrated on the existence of stem cells and
the formation of new neurons in the subventricular zone and in the dentate
gyrus of the hippocampus (Doetsch et al.,
1999; Seri et al.,
2001). In both areas, crucial determinants of neural stem cell
maintenance have been found to lie in the so-called niche, a microenvironment
that provides extrinsic cues driving stem cell proliferation and survival (for
a review, see Alvarez-Buylla and Lim,
2004). However, the essential molecular combination underlying the
adult neural stem cell state, and whether this combination also includes
factors promoting the progenitor state in the embryonic brain, remain
unknown.
To determine the essential mechanisms responsible for the maintenance of
neurogenesis within an adult environment, we need to compare the data
accumulated on the rodent forebrain to other regions of adult neurogenesis
and/or additional vertebrate animal models. To this aim, we focussed our
attention on the zebrafish adult brain, a model system highly appropriate for
the analysis of stem cell maintenance, but for which very few studies have
been conducted. The zebrafish adult brain retains abundant proliferation
activity, and several observations demonstrate that adult neurogenesis in
zebrafish is a true adult phenomenon rather than a persistent embryonic
process. For instance, adult proliferation zones are concentrated in discrete,
usually ventricular, regions (Ekström
et al., 2001; Zupanc et al.,
2005), which suggests the existence of niches as opposed to an
interstitial growth mode. Also, we demonstrated striking molecular and lineage
similarities between adult neurogenesis events in the zebrafish and mouse
adult telencephalon (Adolf et al.,
2006).
In order to identify new stem cell populations using the zebrafish model
system, we considered the mechanisms acting during embryonic development. In
the zebrafish embryo, we previously characterized a pool of progenitor cells
located at the midbrain-hindbrain boundary (MHB). Formation of this pool of
progenitor cells crucially depends on the Hairy/Enhancer of Split [H/E(Spl)]
transcription factors Her5 and Him/Her11, selectively expressed at the MHB and
actively preventing neurogenesis: loss of Her5 or Him/Her11 function leads to
a premature differentiation of primary neurons across the MHB, and
overexpression of her5 inhibits expression of the proneural gene
neurogenin (neurog1/ngn1) and prevents
differentiation in neural plate territories adjacent to the MHB
(Geling et al., 2003;
Geling et al., 2004;
Ninkovic et al., 2005). In
cell lineage tracing experiments, the MHB progenitor pool further proved to be
a dynamic population: over time, cells stop expressing her5 and
him/her11 and exit the pool to enter the adjacent neurogenic regions,
and contribute neurons to the whole midbrain-hindbrain territory
(Tallafuss and Bally-Cuif,
2003). Together, these data implicated Her5 and Him/Her11 as the
key regulators keeping cells in a progenitor state at the embryonic MHB. We
reasoned that such crucial control processes might be reiteratively used over
time to maintain adult neural stem cells, and we searched for the existence of
her5-expressing cells within the adult central nervous system
(CNS).
We show here that her5 is expressed in the adult brain in a restricted ventricular cluster of cells at the MHB, and we demonstrate that these her5-positive cells have all the required properties of stem cells. Together, our results provide, in the genetically tractable animal zebrafish, a new model system for the study of adult neural stem cell biology. They also suggest that the function of H/E(Spl) factors in specifying a neuronal progenitor state might be conserved in adulthood.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), or wild-type zebrafish (Danio rerio) of the AB
strain, were used.
BrdU administration
Fish were injected intraperitoneally with 5 µl/0.1 g body weight of a
freshly prepared bromodeoxyuridine (BrdU) solution at a concentration of 16
mmol/l in 110 mmol/l NaCl pH 7.2. Time to analysis ranged between 3 hours and
8 weeks post-injections. Fish were anaesthetized in tricaine, placed in ice
water for 5 minutes and subsequently decapitated. Brains were dissected and
fixed in 4% paraformaldehyde solution at 4°C for 4 hours, then
progressively dehydrated in methanol and stored in 100% methanol at
-20°C.
Immunohistochemistry
After rehydration of the brains, immunostainings were performed on
vibratome sections (vibrating microtome HM 650 V, Microm): whole brains were
embedded in 3% agarose in PBS and cut serially at 100 µm thickness.
Sections were blocked in PBS with 0.5% Triton X-100 and 10% normal goat serum
for half an hour at room temperature, then incubated in the primary antibodies
at 4°C overnight or 2 hours at room temperature. Primary antibodies used
in this study were rabbit anti-BLBP (1:1500)
(Feng et al., 1994), mouse
anti-BrdU (1:100; Roche), rat anti-BrdU (1:200; abcam), rabbit anti-GFAP
(1:100; DAKO), rabbit anti-GFP TP401 (1:500; ams), mouse anti-GFP (1:200; BD
Bioscience), mouse anti-Hu C/D (1:300; Molecular Probes), mouse anti-PCNA
(1:250; DAKO), mouse anti-PSA-NCAM (1:600; Chemicon), mouse anti-Numb (BD
Biosciences), rabbit anti-MCM5 (1:600, see below). Rb anti-Quacking6 was
kindly provided by Stephane Richard, and rat anti-Musashi was kindly provided
by Hideyuki Okano. Secondary antibodies labelled with Cy2, Cy3 or Cy5 (Jackson
Laboratories) were used at a 1:500 dilution and incubated for 45 minutes at
room temperature. The sections were embedded in Aqua Polymount (Polyscience).
Immunodetection of BrdU required a pretreatment with 2 mol/l HCl followed by
quick washes with borate buffer and PBS. Sections were photographed and
analysed under a Zeiss confocal microscope (LSM 510 META).
Generation of anti-MCM5 antibody
The N-terminal 725 bp of the mcm5 ORF was cloned into the
pTRC-hisB vector (Invitrogen). Proteins were overexpressed in Escherichia
coli using standard procedures and purified using an Ni+NTA
column (Qiagen) under denaturing conditions. Polyclonal antibodies were
produced in rabbits according to standard procedures.
In situ hybridization
In situ hybridization was performed as for whole-mount embryos
(Hauptmann and Gerster, 1994),
starting with whole-mount brains. After hybridization at 70°C and rinses,
brains were embedded in agarose and cut at the vibratome as described above.
Subsequently, blocking, anti-dig incubation and further steps were performed
on free-floating sections. The following ISH probes were used: ash1a
(Allende and Weinberg, 1994),
her5 (Muller et al.,
1996), sox2 [cDNA clone cb236 (B. Thisse, S. Pflumio, M.
Fürthauer, B. Loppin, V. Heyer, A. Degrave, R. Woehl, A. Lux, T. Steffan,
X. Q. Charbonnier and C. Thisse, unpublished)], her4
(Takke et al., 1999),
deltaA (Appel and Eisen,
1998), notch1a
(Bierkamp and Campos-Ortega,
1993). Sections were photographed and analysed under a Zeiss
Axioplan microscope or a Zeiss confocal microscope (LSM 510 META).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Because of the brightness of eGFP, this line allows us to
identify even minute expression zones. We observed two concentrated spots of
Her5-GFP expression in the adult brain, one in each hemisphere
(Fig. 1A,B, arrows),
corresponding to restricted clusters of cells at the intersection between the
valvula cerebelli, torus semicircularis and tegmentum
(Fig. 1D). This location
approximately correlates to the nucleus lateralis valvulae
(Wullimann et al., 1996).
Her5-GFP-positive cells are thus confined to the intersection of two
transition zones: (1) the anterior-posterior (AP) transition separating
cerebellar (valvula cerebelli) from midbrain structures (torus semicircularis,
the equivalent to the mouse inferior colliculus); this location is reminiscent
of the embryonic position of her5-positive cells at the MHB; and (2)
the dorsoventral (DV) boundary between the alar torus semicircularis and the
basal tegmentum, along the tectal ventricle (dotted line in
Fig. 1E-G). Her5-GFP was still
strongly expressed in 2-year-old fish (although in a decreasing number of
cells, data not shown), suggesting that it is in fact maintained over the
animal's lifetime.
Two sets of arguments rule out a potential transgenic artefact and confirm
that the presence of Her5-GFP in the adult brain is the result of
her5 expression at adult stages. First, we examined another
independent transgenic line in which Her5-GFP expression is driven under a 3.4
kb of her5 upstream sequence (-3.4her5:egfp)
(Tallafuss and Bally-Cuif,
2003). Her5-GFP expression was observed at the border between
midbrain and cerebellum in this line as well
(Fig. 1H). Second, we performed
in situ hybridization for the endogenous her5 RNA, and found
her5-expressing cells within the Her5-GFP-positive population
(Fig. 1C-F). Thus, along the
tectal ventricle, Her5-GFP correlates with endogenous her5 expression
and can be used as a valid reporter of her5 gene activation.
Together, these results demonstrate that her5 is expressed throughout
adulthood and characterizes a restricted ventricular cell population at the
adult MHB.
To approach the dynamics of the adult Her5-GFP population, we further
performed a detailed comparison of the expression domains of Her5-GFP and
endogenous her5 RNA on double-labelled preparations. Indeed, while
endogenous her5 and transgenic her5-gfp RNA have an
identical distribution (Tallafuss and
Bally-Cuif, 2003) (data not shown), the stability of Her5-GFP
protein allows its persistence in the cell long after her5 expression
is switched off, and thus permits its use as a tracer of cells that previously
expressed her5. As described, her5 RNA-expressing cells in
the adult brain were concentrated in a cluster directly apposing the
ventricular zone (Fig. 1C,E).
These cells displayed an undifferentiated morphology, with slightly elongated
cell bodies and short processes (Fig.
1G, long arrow, Fig.
2B,C). In addition, we observed that Her5-GFP-positive cells were
present further ventrally, within the tegmentum. The latter cells displayed a
differentiated morphology with rounded cell bodies and long processes
(Fig. 1G-J, small arrows,
Fig. 2F). We conclude that the
Her5-GFP population is dynamic and consists of ventricular cells, located at
the MHB and expressing her5 RNA (and Her5-GFP)
(Fig. 1I,J long arrow), which
give rise to cells located deeper into the tegmentum and maintain only
Her5-GFP (Fig. 1I,J short
arrow).
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;
Tallafuss and Bally-Cuif,
2003). Thus, we asked whether her5-positive cells are
also progenitors in the adult brain. To answer this question, we first
assessed whether her5-positive cells proliferate. The proliferation
markers PCNA and MCM5, which are implicated in the DNA replication machinery
(Maga and Hubscher, 2003;
Ryu et al., 2005) and are
expressed at all phases of the cell cycle, were co-expressed in the same cells
and revealed several proliferation zones in the adult zebrafish brain, in
agreement with previous maps (Adolf et al.,
2006; Zupanc et al.,
2005) (Fig. 2A-F).
Strikingly, however, we found that her5-positive cells were located
within a novel zone of proliferation, which we will refer to as the isthmic
proliferation zone (IPZ). This proliferation zone has also been observed in
another teleost, the stickleback
(Ekström et al., 2001).
The IPZ (Fig. 2A,F-H, yellow
arrows) is neighbouring the tectal proliferation zone
(Fig. 2A,F-H, red arrows),
which has been described in the juvenile and adult medaka fish
(Nguyen et al., 1999), the
juvenile zebrafish (Mueller and Wullimann,
2002) and the stickleback
(Ekström et al., 2001)
and generates cells populating the optic tectum. The two zones are spatially
distinct, and are linked by a narrow ribbon of proliferating cells
(Fig. 2F, white arrow, and not
shown). To determine whether Her5-GFP cells themselves proliferate, we used confocal microscopy to compare the expression of Her5-GFP protein and endogenous her5 RNA with that of MCM5 and PCNA. We observed that in young adults 14% of the Her5-GFP cells located close to the ventricle (n=1175 cells out of eight brains of 2-3 months of age) expressed PCNA. This proportion decreased with age (data not shown). Within the more restricted her5 RNA-positive population, 27% of cells (n=33 cells, two hemispheres) expressed MCM5 (Fig. 2B-E, summarized in Fig. 2H). Hence, about one-third of the her5-RNA-expressing population proliferates, suggesting that these cells might be progenitors.
Adult her5-positive cells are slow proliferating
Because the population of her5-positive cells is located within a
larger population of proliferating cells along the AP axis
(Fig. 2A-E,H), we wondered
whether differences in proliferation rates would define several subgroups of
cells in the IPZ area. In order to answer this question, we assessed the rates
of proliferation in this domain by calculating the labelling index, defined as
the proportion of cells in S-phase within the total proliferating population.
Cells undergoing S-phase were marked by two intraperitoneal injections of BrdU
within a 2-hour interval between injections, followed by the assay after 2
hours. The whole proliferating population was revealed by the expression of
PCNA. The distribution of BrdU- and PCNA-double-positive cells was then
assessed by confocal microscopy and compared to the location of GFP-positive
cells in triple-labelled preparations (Fig.
3). We found that, within the population of ventricular
Her5-GFP-positive cells (Fig.
3A,D green label), only a small proportion of the PCNA-positive
cells (Fig. 3A,C red label)
were in S-phase (Fig. 3B blue
label) at a particular time point (14%, n=390 cells counted in two
brains) (see Fig. 3E,
triple-labelled cells appear white on the merged image, grey arrow). This is
indicative of a long cell cycle for Her5-GFP-positive cells. In striking
contrast, the ventricular domains neighbouring the Her5-GFP zone along the AP
contained a much higher proportion of cells undergoing S-phase (30%,
n=617 cells counted in two brains)
(Fig. 3B,C, examples indicated
by short white arrows). Hence, these domains mostly comprise cells displaying
a shorter cell cycle. These results were confirmed by demonstrating that the
number of cells co-labelled for Her5-GFP and BrdU gradually increased upon
cumulative BrdU labelling (not shown). We conclude that her5-positive
cells are a slow-proliferating population (summarized on
Fig. 3F). The long cell cycle
of her5-positive cells suggests that they could be stem cells in that
region of the brain.
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);
however, available antibodies against these antigens failed to cross-react
with zebrafish. We found several other neural stem cell markers to be
expressed in a subset of her5-positive or ventricular
Her5-GFP-positive cells. For instance, these cells expressed the radial glial
marker BLBP, or the astrocytic and radial glia marker GFAP
(Fig. 4A, parts a-h'),
which usually reflect a neural stem cell state
(Götz and Barde, 2005;
Hartfuss et al., 2001). We
also found a subset of Her5-GFP-positive cells expressing the transcription
factor Sox2 (Ferri et al.,
2004) (Fig. 4A,
parts i-l'), and the asymmetrically inherited cell fate determinants
Musashi (Kaneko et al., 2000;
Okano et al., 2002;
Sakakibara et al., 2002) and
Numb (Petersen et al., 2002)
(Fig. 4B, parts a-h). These
factors have been associated with neural progenitor potential in mammals.
Thus, her5-positive and ventricular Her5-GFP-positive cells possess
molecular attributes of neural stem cells.
her5-positive cells are long-lasting progenitors
These results prompted us to determine whether her5-positive cells
possess the most crucial property of neural stem cells, self-renewal. To do
so, we first labelled S-phase cells with BrdU in a cumulative manner, twice a
day over a time course of 9 days, to increase the number of slow proliferating
cells labelled. This procedure results in labelling roughly 80% of the
PCNA-positive population (not shown). Given that globally 14% of
Her5-GFP-positive cells express PCNA, we estimate that our cumulative BrdU
experiments labelled approximately 12% of the ventricular Her5-GFP-positive
population. Two months after BrdU labelling, the animals were sacrificed and
immunostained for PCNA, BrdU and Her5-GFP, and we searched for long-lasting,
or self-renewing, progenitors (i.e. BrdU-positive cells that maintained PCNA
expression after 2 months). We found that such long-lasting progenitors were
present within the ventricular Her5-GFP-positive population, as shown in
Fig. 5 [the triple-labelled
cell (arrowhead) positive for PCNA (red), BrdU (blue) and Her5-GFP (green)
appears white on the merged image (c); two brains were analysed and seven and
one triple-labelled cells were found, respectively]. More would probably be
revealed if it was technically possible to label the entire slow-proliferating
population. In more frequent cases, we found Her5-GFP-positive cells that had
incorporated BrdU and remained located in the ventricular cell cluster of
neuroepithelial morphology, without expressing PCNA (not shown). These cells,
which do not display a differentiated morphology and are outside the region
expressing differentiation markers (see below), could have entered a quiescent
state. Together, these results show that a proportion of
her5-positive cells possesses the ability to self-renew.
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To determine whether de novo neurogenesis is taking place in the IPZ, we
used two approaches. First, we examined the expression of several proneural
genes involved in the embryonic neurogenic cascade
(Bertrand et al., 2002;
Chapouton and Bally-Cuif,
2004). We observed that deltaA, deltaB, her4, ngn1 and
ash1a were all expressed within or immediately below the IPZ
(Fig. 6A-D, and not shown). At
the cellular level, there was a partial overlap between Her5-GFP and
ash1a or her4 expression (see
Fig. 6C,D). Thus, the molecular
factors required for neuronal differentiation are present in the IPZ area.
Second, we tested whether newborn cells in the tegmentum acquire a neuronal
identity, as revealed by expression of the postmitotic neuronal marker Hu
(Barami et al., 1995;
Mueller and Wullimann, 2002).
To this aim, we labelled progenitors with two pulses of BrdU, and analysed the
identity of their progeny 2 weeks later. We found that, below the IPZ, many
BrdU-labelled cells expressed Hu 2 weeks after their generation
(Fig. 6E-H). Together, these
results demonstrate that de novo neurogenesis is ongoing in the adult IPZ
area.
We next addressed directly the fate of the progeny of the
her5-positive cells, tracing their fate in vivo, using Her5-GFP as a
lineage marker. As described above, Her5-GFP-positive cells that no longer
express her5 transcripts and that display a differentiated morphology
are found within the tegmentum (see Fig.
1G,J, small arrows, Fig.
6L,P,T) after having left the ventricular proliferating pool. To
define the identity of these differentiated Her5-GFP-positive cells, we
studied expression of the neuronal precursor marker PSA-NCAM (a polysialylated
form of the cell surface protein NCAM expressed on young migrating
neuroblasts), the neuron-specific marker Hu, the oligodendrocyte precursor
markers O4 and Quaking (QKI) (Chen and
Richard, 1998; Larocque et
al., 2005) and the astrocytic markers GFAP and S100ß. We
found that few Her5-GFP-positive, her5-RNA-negative cells expressed
O4 and Quaking (Fig. 6I-P, mean
of eight O4-positive cells per brain, analysed in three brains, and five
QKI-positive cells counted in one brain), and thus had acquired an
oligodendroglial fate. These cells were located close to the ventricle in the
anteriormost position within the Her5-GFP cluster. In that same area, we found
few GFP-positive cells expressing S100ß (six cells in one hemisphere of
one brain analysed, data not shown); thus, these cells acquired an astrocytic
fate. In addition, a large proportion of Her5-GFP-positive,
her5-negative cells expressed the neuronal marker Hu
(Fig. 6Q-T). In fact, 100% of
the cells that left the progenitor cluster, displayed long processes and were
located in the tegmentum ventrally to the progenitor pool were Hu-positive
(100 cells per brain, analysed in two brains). Thus, her5-positive
cells also prominently differentiate into neurons in situ. Finally, we also
observed that Her5-GFP-expressing cells located between the ventricular
surface and the Hu-positive neurons expressed PSA-NCAM
(Fig. 4B, parts i-l). In the
mammalian adult forebrain, PSA-NCAM characterizes cells that are still
proliferating but have recently exited the stem cell and transit amplifying
pools to enter the neuronal commitment pathway, and is generally found in
close association with neural stem cell pools
(Doetsch et al., 2002a). Thus,
the co-expression of Her5-GFP and PSA-NCAM demonstrates the existence of the
intermediate, neuroblast step between the her5-positive
stem/progenitor cells and the Her5-GFP-positive, her5-negative,
postmitotic neurons. Together these results demonstrate the multipotentiality
of adult her5-positive cells in their original environment, where
they give rise to differentiated neurons and glia.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A new neural stem cell model in the vertebrate adult brain
The most important finding of the present work is the identification of a
new population of neural stem-cell-like progenitors in the zebrafish adult
brain. Our arguments supporting this conclusion are based on the observations
that cells within the Her5-GFP-positive population: (1) are slow-proliferating
(Fig. 3); (2) are positive for
the expression of the neural stem cell markers BLBP, GFAP, sox2,
Musashi and Numb (Fig. 4); (3)
are self-renewing progenitors (Fig.
5); and (4) give rise to differentiated neurons and glia (Figs
6,
7). These properties together
fulfil the requirements defining the neural stem cell state. Further, we
observed that this population persists throughout life, although, as for
mammalian neural stem cells (Kippin et
al., 2005), the number of her5-positive cells and the
proportion of PCNA-positive cells within the Her5-positive cells tend to
decrease with age (fewer positive cells are found in a 1-year-old brain than
in a 3-month-old brain).
These results are important for several reasons. First, they provide us
with a new model to be compared with known mammalian neural stem cell
populations. Second, this stem cell population is located outside the
telencephalon, further broadening the field of investigation for neural stem
cell characteristics within the adult vertebrate brain. Third, this new model
is established in the zebrafish, a species directly amenable to the study of
gene function by means of transgenes or mutations. Finally, this
stem-cell-like population is readily accessible for experimentation via
Her5-GFP expression in our transgenic model. Together, our findings provide
the stem field with a most promising model to help characterize the general
principles of adult neural stem cell biology. The division and differentiation
potentialities of neural progenitor cells are often addressed in vitro, using
the neurosphere assay (Reynolds et al.,
1992). Using this assay, one conclusive example of
non-telencephalic neural stem cells was recently revealed in mammals at
postnatal stages (Lee et al.,
2005). Because we are working here with a completely new set of
potential stem cells in a new vertebrate model, we were concerned that
alterations in cell potentialities in such culture systems might bias our
findings. Thus we opted for direct, Her5-GFP-mediated tracing of the IPZ stem
cell pool as a more reliable test, providing concomitantly the first
demonstration of a non-telencephalic population of neural stem cells
generating neurons and glia in situ in the vertebrate adult
brain.
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) in
which stem cells are slow-proliferating (the `B' state), and give rise to
committed precursors (the `A' state) via a quickly dividing, transiently
amplifying population (the `C' state). By combining a short BrdU pulse with
PCNA or MCM5 expression studies (Fig.
3), or using cumulative BrdU labelling (not shown), we found,
indeed, that her5-positive cells proliferate slowly. Thus, they probably
represent the `B', true stem cell state. However, it remains unclear whether a
transiently amplifying population originates from the Her5-GFP-positive
domain. We observed that cells adjacent to the her5-positive population along
the AP axis do divide quickly. This is particularly obvious for progenitors
contributing to the growth of the optic tectum
(Fig. 3). However, we could not
detect Her5-GFP expression in these fast proliferating cells. Of particular
importance, within the tegmentum, newborn cells having already acquired the
neuroblast fate (PSA-NCAM) (Fig.
4) or differentiation markers (Hu, Quaking, O4)
(Fig. 6) still express
Her5-GFP. Thus, if progeny cells exit the her5-positive pool along the AP axis
to contribute to the fast-proliferating domain, then their elimination of the
Her5-GFP label must be much faster than that in ventral populations.
Consequently, an abrupt transition would be created from Her5-GFP-positive to
Her5-GFP-negative domains along the AP axis, strikingly different from the
gradual transition observed along the DV axis, where Her5-GFP expression
progressively diminishes towards the ventral tegmentum. Because long-term BrdU
tracing also highlights a ventral migration of ventricular cells of the IPZ
area (Fig. 5B), we currently
favour a model in which ventricular her5-positive progenitors primarily
generate ventral progeny, without a prominent intermediate fast-proliferating
state. This interpretation will, however, need to be tested using direct cell
tracing, independent of the Her5-GFP label.
Another intriguing aspect of the IPZ is the combination of its location at
the MHB and its expression of Her5. We have not directly addressed the origin
of the adult her5-positive pool, and cannot exclude that these cells migrate
into the IPZ from another, possibly neighbouring, source and initiate her5
expression de novo. However, both their final location and their molecular
profile are reminiscent of the embryonic intervening zone (IZ), suggesting
that the IZ and IPZ progenitor pools, in addition to sharing molecular
components, might be related in lineage. In embryos, her5 is expressed
throughout the DV axis; its restriction to the alar-basal boundary in adults
might result from the growth of the dorsally located tectum and torus
semicircularis and the ventrally located tegmentum, leaving a cluster of
expressing cells in between, adjacent to the tectal ventricle. Mammalian adult
neural stem cells of the subventricular zone have also been demonstrated to
originate from a remnant of the embryonic telencephalic ventricular zone, and
their astrocytic nature is also seen as a late derivative of the radial glia,
which serve as neuronal progenitors in the embryo
(Götz and Barde, 2005;
Merkle et al., 2004). Thus,
our observations reinforce the notion that adult neural stem cells might
develop from long-lasting embryonic progenitors. It is clear, however, that
these acquire at adulthood typical characters that contrast them from
embryonic progenitors, such as, at the IPZ, the loss of expression of some
embryonic IZ genes (e.g. him, not shown), the initiation of expression of
glial markers (which are absent from the IZ), and the acquisition of a
slow-proliferation mode.
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) and thus probably to the entire panel of neuronal types in
this region. Whether the adult IPZ is of such functional relevance remains to
be tested, but such experiments are rendered difficult by the fact that the
Her5-GFP label is not permanent. It is lost over time in differentiating
cells, leading to an underestimation of the number of newborn neurons that
actually form from the Her5-GFP-positive zone. Nevertheless, we counted an
average of 100 Hu-positive neurons carrying the transient marker Her5-GFP at a
given time point in the adult tegmentum, and the significance of this value
will be further increased when put in perspective with the size of the
specific neuronal population(s) contributed to by the her5-positive
pool. We have not detected specific cell death in the IPZ area and surrounding
domains within the adult brain (not shown), suggesting that the differentiated
neurons originating from the IPZ are not immediately eliminated on site. The
identification of their neurotransmitter phenotype, as well as of the networks
into which they might be integrated, will await the development of techniques
allowing permanent tracing of her5-positive cells in adulthood.
An important question for future studies will also be to determine whether
the zebrafish IPZ discovered here has a counterpart in adult mammals. Adult
neurogenesis in the mammalian adult substantia nigra was proposed in one study
(Zhao et al., 2003), but these
findings remain controversial (Lie et al.,
2002). Although the substantia nigra belongs to the midbrain, we
believe that it cannot be compared to the zebrafish IPZ, first because these
regions are anatomically distinct (the substantia nigra is located far too
ventrally), and secondly because the zebrafish midbrain is devoid of
dopaminergic differentiation (Rink and
Wullimann, 2001). Because the progenitor properties of the
embryonic IZ have been documented in all vertebrate embryos
(Bally-Cuif et al., 1993;
Hirata et al., 2001;
Palmgren, 1921), and because
it relies, in mammals as well as in zebrafish, on the expression of E(Spl)
factors (Hirata et al., 2001),
our findings here suggest the exciting possibility that a domain equivalent to
the IPZ might also not be restricted to teleosts. Thus it would be most
interesting to re-examine whether the junction between midbrain and hindbrain
in the adult mammalian brain contains stem cells, perhaps using as tools
e(spl) probes. Should this domain in other vertebrates not display a
neurogenic potential, then comparing the developmental steps and the
differential expression genes in the zebrafish and mammalian MHB could also
give crucial insight into the factors involved in the maintenance or
disappearance of this germinal zone.
her5 expression is associated with the maintenance of a stem cell state in the adult brain
We previously demonstrated an active role of Her5 in maintaining the neural
progenitor state during embryonic development. In this study, we now document
expression of her5 in a cluster of progenitors with
neural-stem-cell-like properties in the adult midbrain. These results suggest
that the active role of Her5 in preventing neural differentiation might be
maintained in adulthood, and we are currently manipulating her5
expression within the adult brain to test this hypothesis. A puzzling aspect
of the her5-positive population is its heterogeneity. In particular,
we found within the IPZ her5-positive cells that displayed
neuroepithelial morphology but were PCNA-(or MCM5)-negative
(Fig. 2E,H). Some of them had
incorporated BrdU at an earlier stage (not shown). These cells do not express
differentiation markers; however, they might be in an intermediate state of
commitment towards the differentiated state. Alternatively, and perhaps more
likely as they are located at the ventricle, these cells might have entered a
quiescent state. This interpretation is supported by the fact that PCNA
expression is often shut down or undetectable in quiescent cells
(Maga and Hubscher, 2003), but
will need to be verified once markers of the quiescent state are identified.
Another interesting issue is to determine whether single
her5-positive cells, as opposed to the her5-positive
population, are multipotent in vivo. Such analyses will await the development
of single cell long-lasting tracing techniques in the adult zebrafish brain.
Notably, however, for technical reasons the demonstration of a multipotent
fate at the single cell level in vivo is also still pending for adult
mammalian SVZ stem cells.
A specific class of Notch-independent E(Spl) factors, which act upstream of
the lateral inhibition cascade, is involved in maintaining progenitor pools
within the embryonic neuroepithelium (Bae
et al., 2005; Baek et al.,
2006; Geling et al.,
2003; Hans et al.,
2004; Hatakeyama et al.,
2004; Kageyama et al.,
2005; Ohtsuka et al.,
2006) and our findings suggest that this is extended to the adult
neurogenesis. Interestingly, using the her5pac:gfp transgenic line,
we observed Her5-GFP protein expression in several other very restricted
clusters within the zebrafish adult brain (data not shown). These clusters
were always located in the immediate vicinity of, or within, a proliferating
zone, as for example at the margin of the retina, in the ventricular zone of
the subpallium, in the habenula, or in the hypothalamus. We repeated these
observations in several independent lines (e.g. in -3.4her5:egfp).
Her5-GFP-positive cells were in most regions early postmitotic, and
her5 RNA could not be detected. However, we detected endogenous
her5 expression and the co-expression of PCNA in Her5-GFP-positive
cells within the hypothalamus (not shown). It will now be an important issue
to assess the function(s) of the Notch-independent H/E(Spl) transcription
factor family in adulthood, and determine whether it plays a general role in
maintaining neural stem cells in regenerative regions of adult brains.
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ACKNOWLEDGMENTS |
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