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First published online 18 April 2007
doi: 10.1242/dev.001602
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Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
Author for correspondence (e-mail:
b.appel{at}vanderbilt.edu)
Accepted 20 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: olig2, Neural precursors, Motoneurons, Interneurons, Cell lineage, Lateral inhibition
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) - and these can be subdivided into many distinct
subtypes (Lewis, 2006).
Neuronal diversity is achieved through various developmental strategies. For
example, neural precursors are specified differentially by spatial cues. In
the ventral neural tube, different types of neurons are generated from
distinct neural precursor domains, which are specified at different positions
on the dorsoventral axis by a morphogenic gradient of Sonic hedgehog (Shh)
(Jessell, 2000). Also,
intrinsic signals that change over time can direct the formation of different
neurons from common precursors. The best-described example is that of fly
neuroblasts, whereby temporal transition in the expression of transcription
factors specifies neuronal identity (Brody
and Odenwald, 2002; Pearson
and Doe, 2004). Similar mechanisms could operate in vertebrates.
Foxg1, a winged helix transcription factor, is a key temporal specification
regulator for telencephalic corticogenesis because it suppresses early-born
Cajal-Retzius cell fate in vivo (Hanashima
et al., 2004), and experimental reduction of Foxg1 expression
restores fate plasticity of late cortical progenitors
(Shen et al., 2006).
In addition to spatial and temporal cues, signals that act asymmetrically
in pairs of newly divided sibling cells can specify them for different fates
(Gotz and Huttner, 2005). In
many instances, asymmetric division produces one postmitotic neuron and one
proliferative precursor. However, asymmetric divisions can also produce two
different kinds of neurons. Again, this is best understood in flies where
ganglion mother cells (GMCs) undergo a terminal, asymmetric division to
produce two distinct neurons. For example, the MP neural precursor divides
asymmetrically to produce two different interneurons called dMP2 and vMP2
(Spana et al., 1995).
A critical mechanism for regulating neural cell diversity is cell-cell
communication mediated by Notch receptors and membrane-bound Notch ligands.
Through lateral inhibition, Notch signaling limits the number of cells within
a precursor domain that adopt neuronal fates. In the fly embryonic CNS, loss
of Notch signaling results in the formation of excess neurons at the expense
of epidermal cells (Campos-Ortega,
1995). Notch also influences asymmetric fate decisions. In the
absence of Notch signaling, both daughters of the MP neural precursor develop
as dMP2 (Spana and Doe, 1996).
Thus, in flies, Notch signaling regulates neural fate through at least two
mechanisms. First, through lateral inhibition it regulates the number of cells
specified for neural fate. Second, through binary fate specification it
directs sibling cells for different neuronal fates.
In zebrafish embryos, primary motoneurons that are ablated soon after they
are born are replaced (Appel et al.,
2001), showing that lateral inhibition also operates in
vertebrates to regulate neural development. Numerous lines of evidence
indicate that, as in flies, Notch receptors and their ligands mediate
vertebrate lateral inhibition. For example, expression of dominant-negative
forms of Delta ligands causes formation of excess early-born neurons in frog,
fish and chicken embryos with concomitant decreases in proliferative cells and
later-born neurons and glia (Appel and
Eisen, 1998; Chitnis et al.,
1995; Dornseifer et al.,
1997; Dorsky et al.,
1997; Haddon et al.,
1998; Henrique et al.,
1997). Consistent with these data, zebrafish embryos homozygous
for mutations of mind bomb (mib), which encodes a ubiquitin
ligase necessary for efficient Notch signaling, have excess early-born neurons
and depletion of neural precursors (Itoh
et al., 2003), and Notch mutant mice upregulate expression of
proneuronal transcription factors, indicative of premature and excess neuronal
development (de la Pompa et al.,
1997; Ishibashi et al.,
1995). By contrast, expression of constitutively active forms of
Notch block neuronal development and seemingly maintain neural cells in a
precursor state (Coffman et al.,
1993; Dorsky et al.,
1995; Gaiano et al.,
2000; Park and Appel,
2003). These observations helped foster the view that in
vertebrates Notch diversifies neural fate by determining, through lateral
inhibition, whether a cell differentiates or remains as a precursor
(Gaiano and Fishell, 2002). It
is not yet clear whether Notch signaling mediates asymmetric neuronal fate
specification in vertebrates because the detailed cell lineage information
necessary for identifying binary fate decisions is lacking.
Previously, we showed that a subset of olig2+ ventral
spinal cord precursors produce primary motoneurons and KA' interneurons
at their final division (Park et al.,
2004). Here we describe a series of experiments designed to test
the role of Notch signaling in primary motoneurons and KA' interneuron
specification. Our results provide evidence that Notch signaling operates at
two important levels in vertebrate neuronal specification. First, through
lateral inhibition, it regulates the number of neural precursors that give
rise to specific subsets of neurons. Second, it specifies sibling cells for
different neuronal fates.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
mibta52b mutant fish
(Jiang et al., 1996;
Schier et al., 1996),
Tg(olig2:egfp)VU12
(Park et al., 2004;
Shin et al., 2003),
Tg(hsp70l:XdnSu(H)myc)VU21
(Latimer et al., 2005),
Tg(hsp70:GAL4), Tg(UAS:Notch1aac-myc)
(Scheer et al., 2002) and
Tg(hsp70l:gal4vp16)VU22 fish were used for this study.
Generation of Tg(hsp70l:gal4vp16)VU22 transgenic fish
We first substituted the zebrafish heat shock cognate 70-kd protein,
like (hsp70l) promoter
(Shoji et al., 1998) for the
CMV promoter of pCS2. Next, we inserted gal4vp16 cDNA
(Koster and Fraser, 2001) into
the phsp70l vector, creating phsp70l:gal4vp16. We then
transferred a fragment containing the hsp70l:gal4vp16 fused
to SV40 poly(A) into the pINmega vector
(Latimer et al., 2005), which
contains I-SceI recognition sequences, thus creating
pINmega-hsp70l:gal4vp16. To produce Tg(hsp70l:gal4vp16)
fish, we used the I-SceI-mediated transgenesis strategy
(Thermes et al., 2002) as
described previously (Latimer et al.,
2005). To identify germline transformed founders, G0 fish were
crossed to homozygous Tg(UAS:Notch1aac-myc) fish and the
embryos heat shocked by incubation at 10 hpf for 30 minutes at 36°C.
Tg(hsp70l:gal4vp16);Tg(UAS: Notch1aac-myc)
double-transgenic embryos were identified by morphological defects and
confirmed by anti-Myc immunohistochemistry. To establish stable lines we mated
G0 founders to wild-type fish, raised the embryos to adulthood and screened
them by the same method.
In situ hybridization, immunohistochemistry, BrdU and TUNEL assays
isl2 (Appel et al.,
1995) and her4 (Takke
et al., 1999) antisense RNA probes were generated using
Digoxigenin RNA Labeling Kits (Roche). In situ hybridization was performed as
described previously (Hauptmann and
Gerster, 2000).
For immunohistochemistry, we used the following primary antibodies: mouse anti-BrdU [G3G4, 1:1000, Developmental Studies Hybridoma Bank (DSHB)], anti-c-Myc (Ab-1, 1:100, Oncogene Research Products), mouse anti-HuC/D (1:20, Molecular Probes), mouse anti-Isl (39.4D5, 1:200, DSHB), rabbit anti-phosphorylated histone H3 (1:1000, Upstate Biotechnology) and rabbit anti-GABA (1:1000, Sigma). For fluorescent detection of antibody labeling, we used Alexa Fluor 568 goat anti-mouse, Alexa Fluor 568 goat anti-rabbit conjugate, Alexa Fluor 647 goat anti-mouse conjugate and Alexa Fluor 647 goat anti-rabbit conjugate (all 1:200, Molecular Probes).
For BrdU labeling, manually dechorionated embryos were incubated in BrdU solution (10 mM BrdU and 15% DMSO in EM) for 20 minutes on ice. After washing three times, the embryos were placed in EM, incubated until the appropriate stages at 28.5°C and then fixed with 4% paraformaldehyde. The fixed embryos were immersed in 2 M HCl for 30 minutes and then processed for anti-BrdU and anti-GABA immunohistochemistry at the same time.
For the TUNEL assay, embryos were fixed, dechorionated manually and dehydrated in 100% methanol. The embryos were rehydrated in a graded TBS (10x: 1 M Tris base, 1.5 M NaCl, pH 7.5) series and washed (3x5 minutes) in TBST (1xTBS, 4% Triton X-100). Embryos were rinsed (2x5 minutes) in 1xTTase buffer (5x: 125 mM Tris buffer, 1 M Na cacodylate, 1.25 mg/ml BSA, 1% Tween 20, pH 6.6 at room temperature) without CoCl2. Embryos were preincubated in reaction solution [1xTTase buffer, 1 mM CoCl2, 0.5 µl terminal transferase (Roche), 0.25 µl tetramethyl-rhodamine-5-dUTP (Roche)] on ice, in the dark. Incubation was continued at room temperature for 24 hours. Embryos were washed in TBST (2x5 minutes) followed by the immunohistochemistry procedure.
In situ hybridization images were collected using a QImaging Retiga Exi color CCD camera mounted on a compound microscope and imported into Photoshop (Adobe). Image manipulations were restricted to levels, curve and contrast adjustments. Fluorescence images were collected using a Zeiss LSM510 laser scanning confocal microscope.
Heat-shock induction
To induce expression of the Xenopus laevis dominant-negative form
of Suppressor of Hairless fused to the Myc tag (XdnSu(H)myc), embryos were
collected from matings of Tg(hsp70l:XdnSu(H)myc);Tg(olig2:egfp)
homozygous adults and raised at 28.5°C. At 6 hpf, 9.5 hpf or 11 hpf,
embryos were placed in a prewarmed beaker containing 35 ml of EM in a
39.5°C circulating water bath for 30 minutes and then transferred to fresh
EM and incubated at 28.5°C until fixing stage (20 hpf and 25 hpf).
Similarly, to induce expression of constitutively active Notch1a fused to the
Myc tag (Notch1aac-myc), embryos were collected from matings of
heterozygous Tg(hsp70l:gal4vp16) and homozygous
Tg(UAS:Notch1aac-myc);Tg(olig2:egfp) adults and raised at
28.5°C. At 11 hpf, embryos were transferred to EM either at 34°C (mild
induction) or 36°C (strong induction) for 30 minutes and then returned to
28.5°C until the appropriate stage for fixing. Approximately half of the
embryos should inherit all three transgenes, which we confirmed by
morphological differences, anti-Myc immunocytochemistry and EGFP
fluorescence.
DAPT treatment
DAPT treatments were performed as previously described
(Geling et al., 2002). DAPT
(Calbiochem) was reconstituted with dimethyl sulfoxide (DMSO) to make a stock
concentration of 10 mM. Aliquots were diluted to 100 µM in EM. Embryos were
dechorionated manually and placed in an agarose-coated plate, which contained
the DAPT solution, at 6 hpf. Half of the embryos were washed with EM at 11
hpf. All embryos were returned to 28.5°C until appropriate stage for
fixing. Control embryos were treated with EM containing 1% DMSO only.
Single-cell labeling
Embryos were collected from matings of
mibta52b+/-;Tg(olig2:egfp) adults and raised at
28.5°C. At 10 hpf, embryos were dechorionated manually and mounted, dorsal
side upwards, in 3% methyl cellulose (1500 centipoises, Sigma) submerged in EM
on a depression slide. The slide was mounted on a fixed-stage compound
microscope and viewed using a 40x water-immersion objective. An
injection pipette was backloaded with 2 ml of 5% tetramethyl-rhodamine dextran
(Mr 10,000, Molecular Probes) solution dissolved in 0.2 M
KCl, filled completely with 0.5 M KCl and connected to the electrode, which
was connected to an amplifier. To establish an electrical circuit, a ground
wire was placed in the depression slide. The pipette was positioned on a
single EGFP+ cell and labeled by an electric pulse. 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 24-30 hpf, labeled cells of mutant embryos were analyzed using
a Zeiss LSM510 Meta laser scanning confocal microscope. All clones occupied
the mid-trunk region of the spinal cord (somite levels 6-15).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Notably,
about two-thirds of proximal pMN cells that proliferated produced two distinct
types of neurons, raising the possibility that a binary fate decision
mechanism produces different neurons from some pMN precursors undergoing
terminal divisions. As a first test of this idea, we labeled 11.5
hour-post-fertilization (hpf), five-somite stage Tg(olig2:egfp)
embryos with anti-Isl antibody, which marks PMNs
(Myers et al., 1986;
Park et al., 2002), and
anti-Hu antibody, which labels newly born neurons
(Marusich et al., 1994). This
revealed two types of neurons within the proximal pMN domain, EGFP+
Hu+ Isl+ PMNs and EGFP+ Hu+
Isl- neurons (Fig.
1A). EGFP+ Hu+ Isl- neurons might
be GABAergic VeLD and KA' interneurons because they are born from
olig2+ cells that also produce PMNs
(Park et al., 2004). However,
VeLD and KA' interneurons are not GABA-immunoreactive until about 20 hpf
(data not shown), well after PMNs are evident by gene expression
(Appel et al., 1995;
Inoue et al., 1994;
Tokumoto et al., 1995).
Consequently, we determined the birth date of KA' and VeLD interneurons
to ascertain whether they are born at the same time as PMNs. Beginning at 10
hpf, we pulse-labeled Tg(olig2:egfp) embryos with BrdU at successive
time points and then analyzed BrdU and GABA labeling at 25 hpf. 58% of
KA' cells incorporated BrdU when labeling occurred at 10 hpf
(Fig. 1B,G). The portion of
KA' cells incorporating BrdU fell to 31% at 11 hpf
(Fig. 1C,G) and 7% at 12 hpf
(Fig. 1D,G). No KA' cells
incorporated BrdU when labeling was at 14 and 18 hpf
(Fig. 1E,F,G). These data show
that most KA' precursors undergo a final round of DNA synthesis between
10 and 12 hpf, which correlates with the birth dates of PMNs between 9 and 16
hpf (Myers et al., 1986).
Thus, birth date, gene expression and lineage data are all consistent with the
idea that two different types of neurons can arise from pMN precursors
undergoing terminal division.
mib mutant embryos produce excess PMNs and have a deficit of KA' interneurons
In zebrafish, loss of Notch signaling results in the formation of excess
PMNs (Appel et al., 2001;
Haddon et al., 1998). If Notch
signaling mediates a binary decision between PMN and KA' interneuron
fates, Notch signaling-deficient embryos should have a deficit of KA'
interneurons. To test this, we used gene expression to examine PMNs and
KA' interneurons in embryos mutant for the mind bomb
(mib) gene, which encodes an E3 ubiquitin ligase necessary for
efficient Notch signaling (Itoh et al.,
2003). In the ventral spinal cord of wild-type embryos,
isl2 RNA expression marks 1-2 cells, which are CaP and VaP PMNs, for
an average of about 1.3 cells per hemisegment
(Fig. 2A,E)
(Appel et al., 1995). In
mib mutant embryos, about 4.0 isl2+ cells formed
per hemisegment (Fig. 2C,E). To
count KA' interneurons, we scored EGFP+ GABA+
cells in wild-type Tg(olig2:egfp) and mib;Tg(olig2:egfp)
mutant embryos. Whereas wild-type embryos had an average of 2.1 KA'
interneurons per hemisegment (Fig.
2B,E), mib mutant embryos had an average of only 0.4
KA' cells per hemisegment (Fig.
2D,E). Thus, loss of Notch signaling had complementary effects on
PMNs and KA' interneurons, producing an excess of the former and a
deficit of the latter, consistent with the possibility that Notch signaling
regulates specification of these cell fates in a binary fashion.
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Transient inhibition of Notch signaling during early neurogenesis produces excess PMNs and KA' interneurons
There are at least two, non-mutually exclusive mechanisms by which Notch
signaling might regulate the number of PMNs and KA' interneurons. First,
through lateral inhibition, Notch signaling might limit the number of neural
precursors that undergo a terminal division to produce PMNs and KA'
interneurons. Second, Notch might subsequently regulate a binary fate decision
for PMN and KA' interneuron fate. To discriminate between these
possibilities, we performed two independent series of conditional
loss-of-function experiments of Notch signaling, with the idea that lateral
inhibition and binary fate specification might be temporally separated
processes. First, we used DAPT, a pharmacological inhibitor of
-secretase (Dovey et al.,
2001), which effectively interferes with Notch pathway functions
when applied to zebrafish embryos (Geling
et al., 2002; Latimer et al.,
2005). Whereas DMSO-treated control embryos had averages of 1.3
isl2+ PMNs and 2.1 GABA+ KA' interneurons
per hemisegment (Fig. 4A,B,K),
embryos that were incubated continuously in DAPT solution beginning at 6 hpf
had excess PMNs (average 3.5 isl2+ PMNs per hemisegment)
and a deficit of KA' interneurons (average 0.5 cells per hemisegment) at
20 hpf and 25 hpf, respectively (Fig.
4E,F,K). Thus, continuous DAPT incubation effectively phenocopied
the mib mutant phenotype (compare
Fig. 4A,B,E,F,K with
Fig. 2). Next, we attempted to
inhibit and then restore Notch activity by incubating embryos in DAPT solution
from 6-11 hpf followed by incubation in drug-free embryo medium until 20 hpf
or 25 hpf. To determine if removal from DAPT restored Notch signaling, we
examined expression of the Notch target gene her4
(Takke et al., 1999). At 12
hpf, posterior neural plate expression of her4 was greatly reduced,
relative to control embryos, in embryos treated with DAPT continuously from 6
hpf (see Fig. S2A,B in the supplementary material), whereas her4
appeared to be expressed at intermediate levels in embryos treated with DAPT
from 6-11 hpf, indicating recovery of Notch signaling (see Fig. S2C in the
supplementary material). In contrast to continuous DAPT treatment, DAPT
treatment from 6-11 hpf produced an excess of both PMNs and KA'
interneurons (average 2.3 and 5.3 cells per hemisegment, respectively)
(Fig. 4I,J,K).
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). For
control experiments, non-transgenic embryos were heat shocked at 39.5°C
for 30 minutes or transgenic embryos were raised at normal temperature.
Control embryos showed normal expression of her4 at 11 and 12 hpf
(see Fig. S2D,G in the supplementary material) and had an average of 1.2
isl2+ PMNs and 2.1 KA' interneurons per hemisegment
(Fig. 4K). When heat shocked at
9.5 hpf, Tg(hsp70l:XdnSu(H)myc) embryos did not express her4
at 11 and 12 hpf (see Fig. S2E,H in the supplementary material) and had an
average of 4.1 isl2+ PMNs and 0.5 KA' interneurons
per hemisegment, similar to mib mutant embryos and wild-type embryos
that were continuously incubated in DAPT (compare
Fig. 4C,D with
Fig. 2;
Fig. 4E,F). To inhibit Notch
signaling transiently, as with the DAPT washout experiment, we heat shocked
embryos at 6 hpf, reasoning that the level of XdnSu(H)myc protein would
subside through dilution by cell division or degradation, eventually relieving
Notch signaling inhibition several hours after heat shock. In fact, whereas
transgenic embryos heat shocked at 6 hpf did not express her4 at 11
hpf (see Fig. S2F in the supplementary material), a low level of her4
expression was evident at 12 hpf (see Fig. S2I in the supplementary material),
indicating recovery of Notch signaling. Following heat shock at 6 hpf,
transgenic embryos had an average of 2.4 isl2+ PMNs and
3.5 KA' interneurons (Fig.
4G,H,K), consistent with DAPT treatment from 6-11 hpf. Thus, both
the pharmacological and transgenic experiments show that continuous inhibition
of Notch activity produces excess PMNs and a deficit of KA'
interneurons, whereas transient inhibition during early neural development
results in an excess of both PMNs and KA' cells. These results are
consistent with the possibility that Notch provides two temporally separable
functions in PMN and KA' interneuron development.
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;
Chitnis et al., 1995;
Itoh et al., 2003;
Takke et al., 1999).
Nevertheless, if Notch signaling regulates a binary decision between PMN and
KA' fates, as indicated by the above loss-of-function data, then forced
Notch activity should promote formation of KA' interneurons at the
expense of PMNs. We tested this using a transgenic hsp70l:GAL4VP16
driver to express a constitutively active form of the Notch1a intracellular
domain (Notch1aac). Heat shocking Tg(hsp70l:gal4vp16);Tg(UAS:
Notch1aac-myc) embryos at 36°C for 30 minutes at 11 hpf
produced stronger morphological defects than those previously described using
a hsp70:GAL4 driver (Scheer et
al., 2002) (data not shown). These embryos did not form
isl2+ PMNs or GABAergic neurons (data not shown),
consistent with previous observations that Notch activity blocks neurogenesis
(Itoh et al., 2003;
Takke et al., 1999). Next, we
treated 11-hpf embryos using a milder heat-shock condition of 34°C for 30
minutes to induce transgene expression at an intermediate level. Control
Tg(UAS:myc-Notch1a-intra);Tg(olig2:egfp) embryos formed about 1.3
isl2+ PMNs and 2.1 KA' interneurons per hemisegment
(Fig. 5A,B,G), whereas
Tg(hsp70l:gal4vp16);Tg(UAS: Notch1aac-myc);-Tg(olig2:egfp)
transgenic embryos had an average of 0.2 isl2+ PMNs and
3.7 KA' interneurons per hemisegment, respectively
(Fig. 5C,D,G). By contrast,
Tg(hsp70l:XdnSu(H)myc);-Tg(olig2:egfp) embryos heat shocked at 11 hpf
had an average of 1.9 isl2+ PMNs and 0.5 KA'
interneurons per hemisegment (Fig.
5E,F,G). Thus, activation and inhibition of Notch activity during
the period in which PMNs and KA' interneurons are born have reciprocal
effects on PMN and KA' interneuron number, consistent with a role for
Notch signaling in mediating binary neuronal fate decisions.
Individual neural plate cells produce identical sibling neurons in mib mutant embryos
To investigate the role of Notch signaling at the level of individual
cells, we labeled single EGFP+ neural plate cells of
mib;Tg(olig2:egfp) mutant embryos by iontophoresis at 10 hpf and
determined the identities of clonal descendents at 24-30 hpf by observing
axonal projections. Of ten successful experiments, two labeled cells did not
divide but differentiated as a PMN and VeLD interneuron
(Fig. 6A,B), seven divided once
to produce two neurons each, and one gave rise to three neurons
(Table 1). By comparison, when
we similarly labeled 18 neural plate cells of wild-type embryos, five did not
divide, five produced two neurons and eight gave rise to three to five neurons
(Park et al., 2004). Thus,
clonal size was reduced in mib mutant embryos, consistent with our
proliferation data presented above. Notably, the neuronal composition of
clones was also affected by disruption of Notch signaling. Of the seven
labeled cells that divided once in mib mutant embryos, all produced
two identical neurons, which were either PMNs or VeLD interneurons
(Fig. 6C,E). The three-cell
clone consisted entirely of PMNs (Fig.
6D). In this case, the labeled cell probably divided once to make
a PMN and a cell that divided again to make two PMNs. By contrast, 12 of 13
cells that divided in wild-type embryos produced neurons of different types,
including four of the five cells that divided once
(Park et al., 2004). Together,
these data indicate that Notch signaling acts to extend the proliferative
state of some precursors and, at terminal division, acts to specify sibling
cells for different neuronal fates.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) to test the role of Notch signaling activity in regulating
neural cell specification in zebrafish. Our work establishes that, in addition
to its well-known role as a lateral inhibition signal to limit the number of
precursors that exit the cell cycle and develop as neurons, Notch signaling
specifies sister cells for different neuronal fates in the vertebrate CNS.
The neural plate of frog and zebrafish embryos has been an important model
for investigation of mechanisms that regulate vertebrate neurogenesis. Both
frog and fish embryos produce a relatively small number of early-born primary
neurons, which are first evident within the neural plate. Primary neurons are
organized into three longitudinal bands on either side of the neural plate
midline. PMNs occupy a medial band, interneurons are distributed in an
intermediate band and sensory Rohon-Beard neurons lie within a band near the
lateral edge of the neural plate. Within each band, neurons are intermixed
with proliferative neural precursors in a salt-and-pepper fashion. PMNs that
are ablated before they undergo axogenesis are replaced
(Appel et al., 2001),
suggesting that lateral inhibition regulates allocation of neural precursors
for PMN fate. As in insects, lateral inhibition is mediated by Notch
signaling. Disruption of Notch signaling results in the formation of excess
primary neurons at the expense of precursors and later-born neurons and glia,
whereas forced expression of constitutively active Notch prior to the onset of
neurogenesis blocks neuronal development
(Appel et al., 2001;
Chitnis et al., 1995;
Coffman et al., 1993;
Dorsky et al., 1995;
Henrique et al., 1997;
Park and Appel, 2003). Thus,
Notch, via lateral inhibition, maintains neural cells in a precursor
state.
Although a role for Notch signaling as a lateral inhibitory mechanism is
well established, this is likely to be an incomplete view of Notch function in
vertebrate neural development. A major limitation to a full understanding of
Notch function and cell fate diversification is that the neural cell lineage
remains poorly described. Many models of spinal cord development portray
single types of neurons produced from distinct precursor subdomains aligned on
the dorsoventral axis (Briscoe and Ericson,
2001; Jessell,
2000; Shirasaki and Pfaff,
2002). It is now becoming apparent that each precursor population
produces various cell types. For example, pMN precursors produce
oligodendrocytes in addition to motoneurons
(Richardson et al., 2000;
Rowitch, 2004). Recent
analysis of transgenically marked lineages showed that
Olig2+ pMN cells also give rise to astrocytes and
ependymal cells (Masahira et al.,
2006). Our own analysis of clones resulting from individually
labeled olig2+ precursors in zebrafish showed that these
cells also produce a small number of interneurons
(Park et al., 2004). Thus,
vertebrate neural precursors can give rise to various cell types, raising the
possibility that, as in insects, Notch functions to diversify the fates of
differentiating cells that have a common origin.
Notably, our clonal analysis revealed that some olig2+
precursors produce a PMN and KA' interneuron at their last division
(Park et al., 2004).
Consistent with this observation, we showed that PMNs were often adjacent to
neurons that did not express motoneuron markers during early neurogenesis and
KA' interneurons were born at the same time as PMNs. Formation of excess
PMNs in the absence of Notch signaling is already well documented. Here we
showed that these same embryos had a deficit of KA' interneurons and
that the deficit did not stem from cell death or failure of precursors to
divide. Blocking Notch activity continuously using a pharmacological inhibitor
phenocopied the mib mutation, in producing excess PMNs and a deficit
of KA' interneurons, as did transgenically inducing Notch inhibition to
coincide with the time at which PMNs and KA' interneurons are born. All
these results are consistent with the possibility that Notch signaling
regulates binary neuronal fate decisions. Curiously, embryos in which Notch
signaling was inhibited prior to neurogenesis but active during the time that
PMNs and KA' interneurons were born had an excess of both PMNs and
KA' interneurons. One possible explanation for this result is that
transient Notch inactivation disrupts lateral inhibition, committing more
precursors to lineages that produce PMNs and KA' cells, but restoration
of signaling activity subsequently regulates binary neuronal fate
specification within these lineages resulting in an excess of both cell
types.
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As a final test of the role of Notch signaling in neuronal specification,
we analyzed the clonal descendents of labeled neural plate cells in
mib mutant embryos. Whereas wild-type clones nearly always consisted
of different kinds of neurons (Park et
al., 2004), mib mutant clones always consisted of the
same kinds of neurons. This is similar to the situation in flies, in which MP
neural precursors, which normally produce dMP2 and vMP2 interneurons, form
only dMP2 cells in the absence of Notch activity
(Spana and Doe, 1996).
Additionally, mib mutant clones tended to have fewer total cells than
wild-type clones. Thus, Notch signaling serves both to expand cell number by
maintaining some cells in a proliferative state, and to diversify neuronal
fate by specifying sister neurons for different neuronal identities.
Our observations are most consistent with the model shown in Fig. 7. Within neural precursor populations, lateral inhibitory signaling mediated by Notch receptors and their ligands regulates the allocation of precursors for neuronal fate. In the standard model of lateral inhibition, precursors with high levels of Notch activity remain as precursors, whereas those that have less activity undergo a final division and give rise to neurons. Subsequently, differential Notch activity specifies some olig2+ sibling neurons for different fates. In particular, cells with relatively high Notch activity develop as KA' interneurons, whereas those with lower activity develop as PMNs. Although we focused on just two kinds of neurons, Notch is likely to regulate other kinds of binary neuronal fate decisions. Consistent with this, four of our mib mutant clones consisted of two VeLD interneurons, whereas in wild-type embryos VeLD interneurons share lineages with PMNs and KA' interneurons. Specification of the full range of neuronal fates is likely to require the integration of Notch activity with other signaling molecules such as Sonic hedgehog.
How do sibling neurons have different levels of Notch activity? In flies,
asymmetric distribution of Numb to one of two sibling neurons blocks Notch
signaling, causing the two siblings to take different fates
(Spana and Doe, 1996). Numb
proteins are asymmetrically localized within vertebrate neural cells
(Cayouette and Raff, 2003;
Cayouette et al., 2001;
Shen et al., 2002;
Wakamatsu et al., 1999;
Zhong et al., 1996) and
loss-of-function experiments in mice reveal that they have key roles in
regulating neurogenesis (Petersen et al.,
2002; Petersen et al.,
2004; Zhong et al.,
2000; Zilian et al.,
2001). Lineage analysis performed in cultures revealed that the
formation of morphologically different neurons coincides with asymmetric Numb
distribution (Shen et al.,
2002). In zebrafish, a Numb:EGFP fusion protein distributes
uniformly around the cell membrane of dividing cells during the neural plate
stage (Reugels et al., 2006).
Although this suggests that neuronal fate might not be influenced by
asymmetric localization of Numb, overexpression of fusion protein could swamp
localization signals or Numb might only be asymmetrically distributed to a
small number of cells.
Is binary fate specification a general mechanism for neuronal
diversification in vertebrates? Recent cell lineage-tracing experiments show,
for example, that neuronal progenitors can divide asymmetrically in culture
(Kawaguchi et al., 2004;
Shen et al., 2002), that
zebrafish retinal progenitors expressing ath5 (atoh7 - ZFIN)
divide once to produce a retinal ganglion cell and a distinct postmitotic cell
(Poggi et al., 2005), and that
some dorsal spinal cord dILA and dlLB neurons arise from neuronal progenitors
by asymmetric division in chick (Wildner
et al., 2006). Additionally, elimination of Notch1 signaling in
the mouse retina results in the formation of excess early-born cone
photoreceptors at the expense of other early- and late-born retinal cell types
(Jadhav et al., 2006;
Yaron et al., 2006), which
could reflect a role in mediating binary fate decisions in the retina as we
have shown for spinal cord. Thus, similar to insects, binary fate decisions
regulated by Notch signaling are likely to contribute to the production of
diverse neurons from common precursors in vertebrates.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1911/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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