Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA
Author for correspondence (e mail:
b.appel{at}vanderbilt.edu)
Accepted 22 September 2004
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SUMMARY |
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Key words: Oligodendrocytes, Motoneurons, Hedgehog, Zebrafish, Neural precursor, Spinal cord
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Introduction |
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In amniotes, pMN precursors express Olig1 and Olig2,
which encode basic helix-loop-helix (bHLH) transcription factors
(Lu et al., 2000;
Takebayashi et al., 2000
;
Zhou et al., 2000
) and Olig
gene functions are required for motoneuron and oligodendrocyte development
(Lu et al., 2002
;
Park et al., 2002
;
Takebayashi et al., 2002
;
Zhou and Anderson, 2002
). A
single Olig gene, olig2, is similarly expressed in ventral spinal
cord of zebrafish and necessary for primary motoneuron and OPC formation
(Park et al., 2002
). A
morphogenetic gradient of sonic hedgehog (Shh), which originates from
notochord (mesoderm that underlies the ventral spinal cord) and floor plate
(the ventral-most spinal cord cell type), establishes the Olig expression
domain and, thus, pMN precursors (Jessell,
2000
; Poh et al.,
2002
; Shirasaki and Pfaff,
2002
). A subset of Olig+ cells express
neurogenin (Ngn) genes (Mizuguchi et al.,
2001
; Novitch et al.,
2001
; Park and Appel,
2003
; Zhou et al.,
2001
), which also encode bHLH transcription factors, during the
period of motoneuron production. Ngn expression subsides in ventral spinal
cord cells at about the time that production of motoneurons ends and formation
of OPCs begins (Zhou et al.,
2001
). Additionally, overexpression experiments showed that Olig2
and Ngn2 together can promote motoneuron development
(Mizuguchi et al., 2001
;
Novitch et al., 2001
). These
observations raised the possibility that differential bHLH protein expression
creates a combinatorial code wherein cells that express both Olig2 and Ngn2
develop as motoneurons and those that express only Olig2, following Ngn2
downregulation, develop as OPCs (Zhou and
Anderson, 2002
; Zhou et al.,
2001
). However, the precise mechanisms that regulate specification
of pMN precursors for different fates are not clear.
Here, we address the following questions. First, are pMN cells specified only for motoneuron and OPC fates or do they also give rise to other cell types? Second, can an individual pMN precursor give rise to various progeny or does each precursor produce only a single kind of daughter cell? Finally, what are the molecular signals that promote OPC development from pMN precursors? We show that individual pMN precursors produce a variety of distinct cell types in a manner that is independent of lineage, but spatially and temporally biased. Differences in neural cell response to Hh signaling create these biases, which are necessary for OPC development.
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Materials and methods |
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Single cell labeling
Transgenic embryos at 90% epiboly stage were dechorionated with pronase (5
mg/ml, Sigma) for 10 minutes and rinsed twice with embryo medium (EM)
(Westerfield, 2000). Bud stage
(10 hpf) embryos were mounted, dorsal side upwards, in 3% methyl cellulose
(1500 centipoises, Sigma) submerged in EM on a depression slide. Intracellular
dye labeling was performed essentially as described previously
(Eisen et al., 1989
),
targeting the posterior olig2:EGFP+ domain (see Fig. S1 in
the supplementary material). By inspecting embryos at multiple focal planes
and from different angles, we determined whether one or more cells were
labeled. Greater than 70% of injected embryos were rejected because more than
one cell was filled with dye. Embryos with single labeled cells were
transferred individually to EM with 0.5% penicillin/streptomycin (Gibco) in
24-well plates and raised in the dark at 28.5°C. At 2.5 dpf, labeled cells
were analyzed using a Zeiss LSM510 Meta laser scanning confocal microscope.
All clones occupied positions between somites 6 and 15.
BrdU labeling and immunohistochemistry
Thirty-six hpf embryos were incubated in a 0.5% solution of BrdU (Roche) in
EM for 12 hours at 28.5°C and processed as described previously
(Park and Appel, 2003). For
immunohistochemistry, we used the following primary antibodies: mouse
anti-BrdU (G3G4, 1:1000, Developmental Studies Hybridoma Bank (DSHB), Iowa
City, Iowa, USA), mouse anti-HuC/D (16A11, 1:20, Molecular Probes), mouse
anti-Neurolin (zn-8, 1:1000, DSHB), mouse antizrf1 (1:400, University of
Oregon Monoclonal Antibody Facility), mouse anti-Isl (39.4D5, 1:100, DSHB),
rabbit anti-GABA (1:1000, Sigma). For fluorescent detection of antibody
labeling, we used Alexa Fluor 568 goat anti-mouse conjugate (1:200, Molecular
Probes) and Alexa Fluor 568 goat anti-rabbit conjugate (1:200, Molecular
Probes).
In situ RNA hybridization
In situ RNA hybridization was performed as described previously
(Hauptmann and Gerster, 2000).
Previously described RNA probes included sox10
(Dutton et al., 2001
),
olig2 (Park et al.,
2002
), nkx2.2 (Barth
and Wilson, 1995
), iro3
(Tan et al., 1999
),
shh (Krauss et al.,
1993
) and twhh (Ekker
et al., 1995
). Embryos were sectioned as described previously
(Park and Appel, 2003
). Images
were collected using a QImaging Retiga Exi color CCD camera mounted on a
compound microscope and imported into Adobe Photoshop. Image manipulations
were limited to levels, curves, hue and saturation adjustments.
Cyclopamine treatments
Embryos were incubated in EM containing 100 µM cyclopamine (Toronto
Research Chemicals), diluted from a 10 µM stock in ethanol, at 28.5°C.
To stop the treatment, embryos were rinsed at least three times in EM.
Morpholino injections
The twhh MO, 5'-AAGAGATAATTCAAACGTCATGG-3', has been
described previously (Lewis and Eisen,
2001; Nasevicius and Ekker,
2000
). Approximately 10 nl of a 1 mg/ml solution was injected into
embryos at the one to two-cell stage.
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Results |
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Individual olig2+ cells produce variable, spatially biased lineages
To assess more fully the fates of olig2+ precursors, we
injected individual neural plate cells with vital dye using iontophoresis
(Fig. 2A; see Fig. S1 in the
supplementary material) and identified clonal descendents by morphology at 2.5
dpf (results summarized in Table
1). We could accurately target individual cells because zebrafish
embryos express olig2 RNA during late gastrulation, prior to neuronal
and glial differentiation (Park et al.,
2002), and we can see these cells in living
Tg[olig2:egfp] embryos (Shin et
al., 2003
). At bud to one-somite stage (10-10.3 hpf), cells that
strongly express olig2:EGFP lie in an anteroposterior column about
two to four cells wide posterior to the position of the first somite
(Fig. 2B). These cells overlie
olig2:EGFP- cells of the most medial region of the neural
plate as a result of the morphogenetic cell rearrangements of early
neurulation (Schmitz et al.,
1993
). After neurulation, these cells occupy ventral spinal cord.
We refer to this group of medially located cells as the Proximal/Early domain,
or olig2:EGFPP/E, to reflect the fact that these cells
remain proximal to the floor plate as medial becomes ventral during formation
of the neural tube. Some injected olig2:EGFPP/E cells did
not divide and developed as PMNs (Fig.
2C; Table 1),
consistent with previous observations that PMNs begin to exit the cell cycle
at neural plate stage (Myers et al.,
1986
). Five labeled cells divided once. In a single instance, the
daughter cells were of the same type, with both having ipsilateral axons
projecting toward the head, thus identifying them as KA' cells
(Fig. 2D). In two cases, the
daughters consisted of one PMN and one KA'
(Fig. 2E), and in one case one
daughter was a PMN and the other a VeLD, identifiable by its ipsilateral
descending axon (data not shown). The presence of KA' and VeLD neurons
in our clones is consistent with our above observation that
olig2:EGFP+ cells include ventral GABA+ cells.
The final two-cell clone included a KA' and VeLD
(Fig. 2F). Additionally, eight
clones had from three to five cells, consisting of either PMNs and KA'
cells or PMNs and SMNs (Table
1). Remarkably, no clones contained OPCs.
|
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Motoneurons and OPCs have genetically separable requirements for Hh ligands
The spatial bias evident in our fate-mapping experiments led us to consider
if Hh signaling might play a role in specifying different cell fates within
the olig2 precursor domain. Previous work has described three
zebrafish Hh-related genes and showed that, during early stages of
neural development, notochord expresses shh and echidna
hedgehog (ehh), and floor plate expresses shh and
tiggywinkle hedgehog (twhh)
(Currie and Ingham, 1996;
Ekker et al., 1995
;
Krauss et al., 1993
). In situ
RNA hybridization revealed that at 36 hpf, notochord, medial floor plate and
lateral floor plate cells express shh
(Fig. 4A), whereas only medial
floor plate cells express twhh
(Fig. 4C). Forty-eight hpf
embryos express shh and twhh similar to 36 hpf embryos,
except that lateral floor plate cells express little or no shh RNA
(Fig. 4B,D). Thus, ventral CNS
cells of zebrafish embryos express at least two Hh molecules throughout the
period of motoneuron and OPC specification.
|
|
|
These data show that Hh signaling is required early to initiate the dorsoventral patterning process that forms the olig2+ precursor domain and late to drive olig2 precursors into an oligodendrocyte pathway. To test the idea that Hh signaling also regulates spatiotemporally biased fate specification within the olig2+ precursor domain, we next performed a series of cyclopamine addition and washout experiments. First, we incubated embryos in cyclopamine from 6-26 hpf. When examined at 26 hpf, these embryos did not express olig2 (Fig. 7A) but, instead, expressed iro3 throughout the spinal cord (Fig. 7B, compare with normal pattern in Fig. 6C). These embryos also did not express olig2 (Fig. 7C) or sox10 (Fig. 7D) at 36 hpf and 48 hpf, respectively. Thus, release of Smoothened inhibition after dorsoventral spinal cord pattern was established was not sufficient to recover olig2+ precursors or OPCs. Next, we incubated embryos in cyclopamine from 11-26 hpf. These embryos also did not express olig2 at 26 hpf (Fig. 7E), however, the ventral most spinal cord cells did not express iro3 (Fig. 7F), showing that dorsoventral spinal cord patterning was at least partially intact. In contrast to 6-26 hpf-treated embryos, by 36 hpf ventral spinal cord cells of 11-26 hpf-treated embryos expressed olig2 (Fig. 7G). Thus, in the absence of Hh signaling during this period, embryos maintained a population of spinal cord cells that were competent to express olig2 once they were removed from cyclopamine. A notable difference to untreated embryos is that olig2 was expressed by the ventral-most spinal cord cells of 11-26 hpf treated embryos (compare with Fig. 6A). One explanation is that Hh signaling is continually required to maintain ventral spinal cord precursor domains. Therefore, if Hh signaling is blocked soon after initiation of the dorsoventral patterning process, precursor domains shift ventralwards. Despite the fact that these embryos expressed olig2, they had few OPCs (Fig. 7H; Fig. 8C,D,G) and reduced number of SMNs (Fig. 8G).
|
|
Modulation of Hh signaling can shift the balance between motoneuron and OPC production
To explain the apparent increase in sox10+ OPCs, we
considered the possibility that blocking Hh signaling during 14-26 hpf shifts
olig2D/L precursors from SMN to OPC fate, as this period
coincides with the birth of most SMNs. To investigate this, we repeated the
cyclopamine treatments on Tg[olig2:egfp] embryos and compared OPC and
motoneuron development. Compared with wild-type controls
(Fig. 8A,B,G), embryos treated
from 11-26 hpf had a severe deficit of OPCs
(Fig. 8C,D,G) and fewer SMNs
(Fig. 8C,G), although the
general pattern of neurogenesis, revealed by expression of the pan-neuronal
marker, Hu, appeared normal (Fig.
8C). By contrast, embryos treated from 14-26 hpf had a 1.8-fold
excess of OPCs and a 1.7-fold decrease in secondary motoneurons compared with
wild type (Fig. 8E-G). The
complementary increase and decrease in these cell types strongly suggests that
a temporary block of Hh signaling can redirect precursors that would normally
develop as SMNs for OPC fate.
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Discussion |
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Spatial and temporal roles for Hh signaling in patterning theolig2+ precursor domain
An obvious candidate for regulating formation and patterning of the
olig2+ precursor domain is the Hh signaling system. The
idea that a morphogenetic gradient of Shh establishes distinct precursor
domains in the ventral spinal cord is well established
(Jessell, 2000;
Lee and Pfaff, 2001
). However,
whether Hh signaling also regulates fate specification within a precursor
domain has not been explored. Our work now shows that Hh signaling is
necessary at multiple steps during neural development to specify
oligodendrocytes from precursors that also produce motoneurons and
interneurons (Fig. 9).
|
Once Hh signaling induces olig2 expression within a subset of
spinal cord precursors, it is required continuously during early stages of
neurogenesis to maintain its expression. We showed that embryos treated with
cyclopamine at 11 or 14 hpf, after initiation of olig2 transcription,
no longer expressed olig2 by 26 hpf. However, if they were then
removed from the cyclopamine, by 36 hpf they re-expressed olig2, but
in a domain ventral to the normal position of olig2+
cells. These in vivo results are similar to work that showed that ventral
neural plate explants cultured in the absence of Shh re-expressed Pax7, a
marker of dorsal spinal cord, whereas slightly older ventral neural explants
did not (Ericson et al.,
1996). Together, these data suggest that during early stages of
neural development, neural cells are plastic and require an extended period of
exposure to Hh signals to stabilize ventral precursor domains.
We also found that the entire olig2+ precursor domain
appears to be formed dynamically by continuous exposure to Hh signals rather
than all at once (Fig. 9). Our
fate mapping showed that olig2:EGFP- cells that border
olig2:EGFP+ cells in the neural plate in wild-type embryos
at 10 hpf later express the transgene and it is specifically these cells that
produce many of the secondary motoneurons and all of the OPCs. Our functional
data suggest that Hh signaling promotes expansion of the
olig2+ domain and OPC production. First,
syu-/- embryos express olig2 but they have fewer
than normal secondary motoneurons and almost no OPCs.
syu-/- embryos were previously shown to have appropriate
numbers of primary motoneurons (Lewis and
Eisen, 2001), which we show originate within the
olig2P/E domain. One possible interpretation of these
observations is that Shh is required for formation of the
olig2D/L domain but not the olig2P/E
domain. Second, embryos treated with cyclopamine from 14-26 hpf had dorsally
enlarged olig2+ domains relative to those treated from
11-26 hpf and only the 14-26 hpf group produced OPCs. We interpret our data to
mean that, in zebrafish, exposure to Hh between 11 and 14 hpf is crucial for
expanding the olig2P/E domain to include the
olig2D/L domain, which gives rise to OPCs. We conclude
that although all pMN precursors express olig2, they do so at
different times and that this correlates with a spatial bias in cell fate.
Thus, time and position dependent specification of olig2+
precursors by Hh signaling contributes to cell fate diversification
(Fig. 9).
Subsequent to formation of the olig2+ domain, a
differential response of olig2D/L cells to Hh signals
apparently determines whether they develop as SMNs or OPCs. In particular, if
we blocked Hh signaling with cyclopamine from 14 hpf-26 hpf, during the period
of SMN birth, we later found that these embryos had deficits of SMNs,
suggesting that continuous exposure to hedgehog proteins is necessary to drive
an olig2+ cell into that differentiation pathway.
Similarly, by applying recombinant Shh and function-blocking anti-Shh
antibodies to neural explants, Ericson et al.
(Ericson et al., 1996)
produced evidence interpreted to mean that Shh first ventralizes the spinal
cord and then later (until late S-phase prior to cell cycle exit) promotes
motoneuron development from ventralized precursors. However, these experiments
did not establish the fate of ventralized precursors from which Shh was then
removed by function-blocking antibody. Our cyclopamine data show that
secondary motoneuron deficits were accompanied by complementary increases in
OPC number, if Hh signaling was restored after the period of motoneuron
development. Thus, differential response of olig2+
precursors to Hh signals could specify them as motoneurons or OPCs. Recent
work described methods to promote formation of motoneurons from embryonic stem
cells in culture (Wichterle et al.,
2002
). Our results raise the possibility that modulation of Hh
signaling could enrich the production of oligodendrocytes from the same
cultures.
Finally, our data provide strong evidence that Hh signaling is required for
OPC specification in a manner independent from its role in spinal cord
dorsoventral pattering. Similar to function blocking anti-Shh antibody
experiments (Orentas et al.,
1999; Soula et al.,
2001
), our cyclopamine experiments showed that Hh signaling is
necessary until the time that OPCs normally appear, well after dorsoventral
pattering is completed. These data favor the idea that late Hh signaling plays
a direct role in OPC specification rather than an indirect one, through
induction of a secondary signaling pathway. As this late requirement is
similar to a late requirement for Hh signaling in motoneuron specification
(Ericson et al., 1996
), our
results raise the possibility that Hh acts as a general signal to drive
olig2+ cells into a differentiation pathway.
Recently reported work has provided evidence that the timing of Hh
signaling plays an important part in specifying distinct muscle cells in
zebrafish (Wolff et al.,
2003). Similarly, different responses to Hh over time appear to
contribute to patterning of the telencephelon
(Kohtz et al., 1998
). Our own
studies now show that the variety of cell types that arise from a common
ventral spinal cord precursor population is rich and modulation of Hh
signaling can influence the kinds of cells that this population produces.
Thus, Hh not only functions as a morphogen to establish a precursor domain,
but also subsequently contributes to diversification of cell fate within
it.
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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/131/23/5959/DC1
* These authors contributed equally to this work
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