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First published online August 25, 2008
doi: 10.1242/10.1242/dev.020479
Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Department of Zebrafish Neurogenetics, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, D-85764 Neuherberg, Germany.
Author for correspondence (e-mail:
bally{at}helmholtz-muenchen.de)
Accepted 16 July 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: E(Spl), Her5, Her11, Zebrafish, Midbrain-hindbrain boundary, Neural progenitor, Gsk3β, PKA, Gli1
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kageyama
et al., 2005; Panchision and
McKay, 2002). To approach these mechanisms, we are focusing on an
evolutionarily conserved progenitor pool, the `intervening zone' (IZ), which
adjoins the midbrain-hindbrain boundary (MHB) in the neural tube of all
vertebrate embryos (Bally-Cuif et al.,
1993; Geling et al.,
2003; Stigloher et al.,
2008; Vaage, 1969)
(Fig. 1A-C). Lineage tracing of
the IZ pool in the zebrafish embryo demonstrates that it progressively
contributes neurons to the entire midbrain-hindbrain domain (MH)
(Tallafuss and Bally-Cuif,
2003), a territory involved in a number of physiological and
integrative functions, including sensory processing, motor control and social
behavior.
The multiple functional outputs of the MH require a diversity of neuronal
subtypes organized in a precise neuroanatomical pattern. Two properties of the
IZ demonstrate its importance in organizing MH maturation. Along the
anteroposterior (AP) axis, the temporal order with which cells leave the IZ to
populate the differentiating MH correlates with the future spatial
organization and subtype of the neurons that they generate
(Tallafuss and Bally-Cuif,
2003). Hence, the mechanisms controlling maintenance of the IZ
along the AP axis are likely to influence the generation of MH neuronal
subtypes. Along the dorsoventral (DV, initially mediolateral) axis, IZ cells
exhibit differences in their propensity to undergo neurogenesis, which
correlate with the earlier maturation of the basal plate as compared with the
alar plate (Easter et al.,
1994): at early neurogenesis stages, medial (future ventral) IZ
(MIZ) cells are more prone to undergo neurogenesis than lateral (future
dorsal) IZ (LIZ) cells (Ninkovic et al.,
2005).
The maintenance of IZ progenitors is controlled by E(Spl) bHLH
transcription factors, such as mouse Hes1 and Hes3 or zebrafish Her5 and
Her11, which inhibit expression of proneural genes, such as neurogenin
1 (neurog1) (Hatakeyama et
al., 2004; Kageyama et al.,
2005; Stigloher et al.,
2008). The compound genetic ablation of mouse Hes1 and
Hes3 leads to the premature differentiation of the IZ and loss of MH
neuronal identities (Hatakeyama et al.,
2004; Hirata et al.,
2001). her5 and her11 are expressed across the
IZ and play redundant roles in IZ maintenance
(Ninkovic et al., 2005).
Specifically, the IZ is sensitive to a total level of Her5+Her11, with
differences along the mediolateral axis: three copies of her5 and/or
her11 are sufficient to maintain the IZ, two copies are enough to
maintain the LIZ but not the MIZ (which transforms into a
neurog1-positive zone) (Fig.
1B), and a lower amount leads to ectopic expression of
neurog1 across the LIZ as well
(Ninkovic et al., 2005)
(Fig. 1C). Hence, E(Spl)
activity controls IZ maintenance and is sensed differently by MIZ and LIZ
cells.
Unlike classical E(Spl) genes, her5 and her11 are not
activated by Notch signaling (Bae et al.,
2005; Geling et al.,
2004; Hans et al.,
2004; Hatakeyama and Kageyama,
2006; Stigloher et al.,
2008), and the molecular cascades involving E(Spl) activity during
IZ formation remain unknown. Similarly, the mechanisms rendering MIZ cells
less sensitive to E(Spl) activity than LIZ cells need to be discovered. To
reveal these mechanisms, we used sensitized conditions in which the total
level of E(Spl) activity is reduced, and we tested the influence of several
signaling cascades and signal transduction pathways active in the anterior
neural plate. We show that the kinases Gsk3β and PKA are novel
determinants of IZ formation in zebrafish, and demonstrate that Gsk3β/PKA
cooperate with E(Spl) activity in a dose-dependent manner throughout the IZ.
Similar to E(Spl) factors, Gsk3β and PKA activities are sensed
differentially along the mediolateral axis. We demonstrate that the
transcription factor Gli1, expressed within the MIZ and displaying neurogenic
activity, accounts for this differential response. Surprisingly, we also show
that this activity of Gli1 is independent of Hedgehog (Hh) signaling. Our
results provide a molecular framework to help understand IZ maintenance and
its properties along the AP and DV axes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
Tg(her5PAC:EGFP)ne1939
(Tallafuss and Bally-Cuif,
2003)] fish were staged according to Kimmel et al.
(Kimmel et al., 1995).
smoothened (smub641)
(Varga et al., 2001) and
Dfw5/w5 (Lekven et
al., 2003) mutants were obtained by pairwise mating of
heterozygous adult carriers.
In situ hybridization and immunocytochemistry
In situ hybridization experiments were performed as described
(Hammerschmidt et al., 1996b;
Ninkovic et al., 2005) with
the following probes: her5
(Müller et al., 1996),
neurog1 (Korzh et al.,
1998), pax2.1 (pax2a - ZFIN)
(Lun and Brand, 1998),
gli1 [recloned using the primers recommended by Thisse and Thisse
(Thisse and Thisse, 2005) for
probe n°eu934], gli2
(Karlstrom et al., 2003),
gli3 (Tyurina et al.,
2005), wnt1 (Molven
et al., 1991), myoD (myod1 - ZFIN)
(Weinberg et al., 1996),
shh (Krauss et al.,
1993) and EGFP (Clontech). Primary antibodies for
immunohistochemistry were rabbit anti-GFP (AMS Biotechnology Europe, TP401,
1/500), mouse anti-human neural protein HUC/HUD (ELAVL3/4 - HUGO) (MoBiTec,
A-21271, 1/300), rat anti-BrdU (Abcam, 1/200) and rabbit anti-phospho-histone
H3 (Upstate Biotechnology, 1/200), revealed using FITC-, AF488- or
Cy3-conjugated secondary antibodies (Invitrogen). BrdU immunohistochemistry
involved pretreatment with 3.3 M HCl for 30 minutes at room temperature,
followed with two 15-minute washings in sodium tetraborate buffer (0.1 M, pH
8.5). Embryos were scored and photographed under a Zeiss Axioplan
photomicroscope.
RNA, morpholino and gripNA injections
Capped RNAs were synthesized using the Ambion mMessage mMachine Kit
following the supplier's instructions and were injected at the one-cell stage:
a constitutively active form of protein kinase A, PKA*
(Hammerschmidt et al., 1996a)
at 20 ng/µl; a dominant-negative form of the PKA regulatory subunit,
dnReg (Strähle et al.,
1997) at 50 ng/µl; and truncated Patched
(patched
loop2)
(Briscoe et al., 2001) at
50-150 ng/µl. Morpholino antisense oligonucleotides (gli1MO)
(Gene-Tools, OR) and gripNA antisense oligonucleotides (her11GripNA
and her5GripNA) (Active Motif, Belgium) were injected into one-cell
stage embryos at 0.5 mM. Sequences of antisense oligonucleotides are:
her5GripNA, 5'-GGTTCGCTCATTTTGTG-3';
her11GripNAATG, 5'-ATTCGGTGTGCTCTTCAT-3'
(Ninkovic et al., 2005); and
gli1MO, 5'-CCGACACACCCGCTACACCCACAGT-3'
(Karlstrom et al., 2003).
Pharmacological treatments
Glycogen synthase kinase 3 β (Gsk3β) inhibition was achieved by
applying LiCl or Gsk3β inhibitor III
(2,4-dibenzyl-5-oxothiadiazolidine-3-thione, OTDZT) (Calbiochem, Germany).
Lithium (0.3 M LiCl) treatments were performed at 28°C in embryo medium
for 15 minutes, followed by three washes in embryo medium and further
development. OTDZT was diluted to 1 mM in embryo medium and applied as with
LiCl. Cyclopamine (BIOMOL, Germany) was applied at 100 µM to dechorionated
embryos from the stages indicated until fixation for in situ hybridization.
Controls were soaked in carrier only.
Live confocal imaging of MIZ and LIZ precursor cells
Tg(her5PAC:EGFP)ne1939 transgenic zebrafish embryos
(Tallafuss and Bally-Cuif,
2003) were injected with 50 ng/µl 2xlckmRFP capped RNA
(Megason and Fraser, 2003) in
two central cells at the eight-cell stage. Developing embryos were imaged live
using an inverted laser-scanning confocal microscope (LSM510Meta, Zeiss) as
described (Distel and Köster,
2007; Köster and Fraser,
2004). Development was followed at 28°C using a 20x 0.5
Plan-Apochromat objective from shield until tailbud stage, when distinct
expression of eGFP was visible. Three-dimensional stacks were collected every
5 minutes using excitation at 561 nm to record 2xlckmRFP protein. The movies
were stopped when the embryos reached 90% epiboly and the final six scans were
continued manually, including excitation at 488 nm to record GFP protein and
locate the IZ. Subsequent image processing was performed using LSM software
(version 3.2 SP1.1, Zeiss) and Photoshop (version 9.0, Adobe Systems).
Tracking of cells was performed using the manual tracking function in ImageJ
(version 1.37a, Wayne Rasband, National Institutes of Health USA,
http://rsb.info.nih.gov/ij/)
with the plug-in from Fabrice Cordeli (Institut Curie, Orsay, France).
BrdU labeling
Wild-type embryos were dechorionated and soaked in 10 mM BrdU, 15% DMSO in
embryo medium for 10 minutes on ice. Embryos were then washed three times on
ice for 10 minutes each with washing buffer containing 15% DMSO, fixed
immediately and processed for BrdU immunohistochemistry.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lekven et al.,
2003). Because Wnt signaling has been implicated in controlling
neurogenesis in other instances (e.g.
Megason and McMahon, 2002;
Panhuysen et al., 2004;
Zechner et al., 2003), in
particular by supporting the proliferating state, we tested whether it
influenced MIZ and LIZ cells. We expected that Wnt would favor the progenitor
state and that higher Wnt signaling laterally would account for the higher
sensitivity of LIZ cells to the inhibition provided by Her5/Her11.
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;
Klein and Melton, 1996)
affected IZ formation. Wild-type embryos were incubated in 0.3 M LiCl for 15
minutes at late gastrulation, then washed and allowed to develop until the 3-
to 5-somite stage, when they were analyzed for neurog1 expression.
When LiCl was applied at 60% epiboly, the anterior neural plate was
posteriorized, as in mutants with hyperactivated Wnt
(Dorsky et al., 2003;
Heisenberg et al., 2001;
Kim et al., 2000;
van de Water et al., 2001)
(Fig. 2A). The IZ was enlarged
along the AP axis, but neurogenesis inhibition was not affected (90% of cases,
n=50) (Fig. 2A). By
contrast, when LiCl was applied at 80% epiboly, prominent expression of
neurog1 was observed ectopically within the MIZ (83% of cases,
n=60) (Fig.
2B,E,E', arrows, compare with
Fig. 2D,F,F'), with only
a moderate effect on AP patterning. This neurogenic phenotype was crucially
dependent on the stage of LiCl application: incubations at later times did not
affect IZ formation (88% of cases, n=56)
(Fig. 2C). Thus, LiCl applied
at 80% epiboly induces the premature commitment of MIZ progenitors towards
differentiation.
Because LIZ cells are less prone to undergo neurogenesis than MIZ cells, it
is possible that LiCl modulates neurogenesis throughout the IZ, but that its
effects are only visible medially in the wild-type embryo. To test this, we
further applied LiCl to embryos in which Her5 activity was decreased by
injection of her5 antisense oligonucleotides (her5GripNA).
Here, neurog1 expression was induced by LiCl throughout the IZ (86.5%
of cases, n=52) (not shown), a phenotype never obtained with
her5 knockdown alone (Fig.
1B) (Ninkovic et al.,
2005). Thus, in the presence of LiCl, IZ cells are globally
rendered more susceptible to undergo neurogenesis. her5 or
her11 expression was not modified by LiCl treatment (96% of cases,
n=30) (not shown). LiCl might rather modulate Her5/Her11 protein
activity, or act downstream or in parallel to these factors.
Importantly, the effect of LiCl is opposite to that expected if endogenous
Wnt signaling controlled the behavior of MIZ and LIZ cells: the expression of
Wnt ligands in the LIZ would predict that activated Wnt decreases the tendency
of a cell to differentiation. Hence, the neurogenic effects of LiCl might not
be linked to Wnt signaling activation. To directly test this interpretation,
we assessed the effects of LiCl on blockade of canonical Wnt downstream of
LiCl activity. We used hsp70l:tcf3-GFPw26 transgenic
embryos, which express a truncated form of Tcf3a (Tcf7l1a - ZFIN) that acts as
a dominant repressor of canonical Wnt upon heat shock
(Lewis et al., 2004). We
submitted these embryos to a heat-shock pulse at 50% epiboly, followed 2 hours
later by LiCl treatment (at 80% epiboly), and analyzed neurog1
expression at the 3- to 5-somite stage. A 2-hour delay after the heat-shock
pulse is sufficient to significantly antagonize Wnt activity at gastrulation
(Lewis et al., 2004); thus,
Gsk3β inhibition by LiCl should occur while downstream components of
canonical Wnt signaling were blocked. neurog1 expression was still
strongly induced by LiCl across the MIZ (83% of cases, n=42)
(Fig. 2, compare G with H),
showing that LiCl is unlikely to influence IZ neurogenesis via activation of
Tcf-mediated Wnt signaling.
|
), we were
unable to reveal active canonical Wnt signaling in the anterior neural plate
at the onset of neurogenesis (98% of cases, n
100), whereas
GFP expression was obvious in active Wnt signaling domains such as
the embryonic margin (not shown). Secondly, lowering Wnt signaling (e.g. in
hsp70l:tcf3-GFPw26 transgenic embryos heat-shocked at 50%
epiboly) did not change neurog1 induction, neither by itself nor upon
Her5 blockade (not shown). We conclude that canonical Wnt signaling is not
involved in modulating the tendency of IZ cells to undergo neurogenesis, and
that LiCl affects IZ neurogenesis independently of canonical Wnt.
LiCl effect on neurogenesis control is mimicked by inhibiting Gsk3β
LiCl effects are broad-ranged, but primarily inhibit Gsk3β activity
(Berridge et al., 1989;
Hedgepeth et al., 1997;
Klein and Melton, 1996). To
confirm that LiCl-induced neurogenesis across the IZ was mediated by
Gsk3β blockade, we tested the effects of the selective Gsk3β
inhibitor OTDZT (Martinez et al.,
2002). Similar to LiCl treatment, wild-type embryos were incubated
in OTDZT at 80% epiboly for 15 minutes, and processed for neurog1 in
situ hybridization at the 3- to 5-somite stage. OTDZT induced ectopic
neurog1 expression across the MIZ, mimicking the LiCl effect (87% of
cases, n=38) (Fig.
2I-J'). Furthermore, as with LiCl, lowering Her5/Her11
dosage revealed that Gsk3β is active throughout the IZ (85% of cases,
n=40) (Fig. 2K,L).
Therefore, Gsk3β is a crucial element modulating neurogenesis at the IZ
and is required in vivo for the formation and early maintenance of the
MIZ.
Activated PKA compensates for Gsk3β inhibition or reduced E(Spl) activity
In addition to targeting canonical Wnt, Gsk3β is involved in a number
of signaling pathways, where it triggers enhanced phosphorylation of target
proteins after these have been primed by phosphorylation via cAMP-dependent
protein kinase A (PKA) (Jia et al.,
2002; Price and Kalderon,
2002; Zhang et al.,
2005). To assess whether such a process might be at play during IZ
formation, we tested whether PKA activation influences the neurogenic effect
of inhibiting Gsk3β. As reported, only a few embryos (50% in our case)
injected with capped mRNA encoding a constitutively active catalytic subunit
of PKA (PKA*) (Hammerschmidt et
al., 1996a) develop a normal neural plate
(Blader et al., 1997). Among
these, all formed a normal IZ (Fig.
3, compare A,F with D,H). However, PKA* inhibited the
neurogenic effect of LiCl, restoring the MIZ when LiCl was applied to
PKA*-injected embryos (88% of cases, n=61)
(Fig. 3, compare C with B).
Therefore, general activation of PKA does not in itself expand the IZ, but it
can compensate for the loss of Gsk3β function to maintain neurogenesis
inhibition in this location.
We next addressed whether PKA activation could also compensate for a downregulation of E(Spl) function at the MHB, by testing the effects of co-injecting her5GripNA and PKA* capped RNA into wild-type embryos. Whereas blocking Her5 function with gripNAs lead to a loss of the MIZ in the vast majority of cases (92% of cases, n=26) (Fig. 3E), the co-expression of PKA* efficiently rescued this phenotype and restored MIZ formation (66% of cases, n=50) (Fig. 3, compare G with H). Thus, general activation of PKA also promotes neurogenesis inhibition downstream of, or in parallel to, Her5 function.
Activated PKA and Gsk3β act in concert with E(Spl) factors to permit IZ formation
To determine whether PKA is in itself required for IZ formation, we blocked
its activity by injection of capped RNA encoding a dominant-negative form of
the PKA regulatory subunit, dnReg
(Strähle et al., 1997).
dnReg robustly prevents the transcription of downstream targets of the Hh
pathway, such as spalt (sall1a - ZFIN), that require PKA for
their expression (88% of cases, n=72) (not shown). In embryos
expressing dnReg, the MIZ was replaced by ectopic neurog1 expression
(84% of cases, n=50) (Fig.
4, compare C with A). When Her5 activity was also inhibited by the
co-injection of her5GripNA, ectopic neurog1 expression was
also detectable within the LIZ (86% of cases, n=35)
(Fig. 4, compare E with A-C).
To demonstrate that these cells were truly induced within the LIZ and did not
originate from the neurog1-positive r2L domain (see
Fig. 1A), we counted the number
of cells in r2L upon co-injection of her5GripNA and dnReg.
This was unchanged as compared with control embryos
(Fig. 4I). Cell proliferation
in this domain, measured by the number of cells expressing the M-phase marker
phospho-histone H3 (PH3), also remained comparable to control embryos
(Fig. 4J). Hence, migration of
precursors from the r2L area, possibly compensated for by increased
proliferation in r2L, is unlikely to account for the ectopic
neurog1-positive cells found within the LIZ. Rather, we conclude that
neurog1 expression is ectopically induced in this location upon
blocking Her5 and PKA activities. Thus, blocking PKA increases the propensity
of all IZ cells to undergo neurogenesis, and PKA is crucially required in vivo
for MIZ formation.
|
Together, these results place neurogenesis inhibition by Gsk3β/PKA
downstream of, or in parallel to, E(Spl) activity. To resolve this issue, we
tested whether increased E(Spl) activity would compensate for decreased
Gsk3β/PKA. We blocked PKA in Tg(her5PAC:EGFP)ne1939
transgenic fish (Tallafuss and Bally-Cuif,
2003), which carry one additional copy of her11 under the
control of its own regulatory elements, and thereby express three doses of
Her5/Her11 activity in a correct spatio-temporal manner
(Ninkovic et al., 2005). This
transgenic background permits normal MIZ formation in the absence of Her5
(Ninkovic et al., 2005).
However, it proved insufficient to block ectopic neurog1 activation
and rescue the MIZ upon expression of dnReg (79% of cases, n=56)
(Fig. 4, compare H with G).
dnReg had no effect on the level of expression of her5 and
her11, and we failed to detect putative PKA or Gsk3β
phosphorylation sites on Her5 and Her11 (not shown). Although we cannot
exclude the possibility that higher doses of Her5/Her11 could be effective,
the most parsimonious explanation for these results is that Gsk3β/PKA act
downstream of E(Spl) factors.
MIZ and LIZ cells differ intrinsically in a cell cycle-independent process
Cell-intrinsic components or local signaling cues could account for
rendering the MIZ and LIZ different in their response to the Gsk3β/PKA
and Her5/Her11 pathways. To test for the relevance of cell-intrinsic
mechanisms, we first assessed whether MIZ and LIZ differ in lineage. Extensive
cell exchanges across the midline of the zebrafish embryonic neural tube have
been documented, as well as dorsoventral dispersion, after the 2-somite stage
(Papan and Campos-Ortega,
1997), but not before (Woo and
Fraser, 1995). However, IZ precursors have not been specifically
studied in this context. To address this, we time-lapsed MIZ and LIZ
precursors from the shield stage onwards using confocal microscopy in
Tg(her5PAC:EGFP)ne1939 transgenic embryos, in which
her5-expressing cells are also positive for GFP
(Tallafuss and Bally-Cuif,
2003). With the help of a lineage reporter (2xlckmRFP)
(Megason and Fraser, 2003)
injected as capped RNA into a subset of cells, by playing the movies backwards
from the tailbud stage we could trace the origin of single cells that
contribute to the MIZ and LIZ (as well as to the r2M and r2L domains). We
observed that these precursor cell groups remained spatially segregated at all
times between the shield and tailbud stages
(Fig. 5A,B). In particular, we
did not observe any contribution of MIZ precursors to the LIZ and vice versa.
This raises the possibility that MIZ and LIZ precursors inherit different
determinants that might influence their sensitivity to neurogenesis
inhibitors.
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),
we tested whether MIZ and LIZ cells differed in their proliferation
characteristics. Markers for the S (BrdU incorporation) and M (PH3, and
mitotic figures) phases were analyzed together with the IZ marker
her5 in triple-labeled preparations at 75% epiboly to tailbud. At
these stages, there is still a tendency for cells to divide synchronously
(Kimmel et al., 1994) and thus
the co-analysis of S- and M-phase markers on single specimens was important to
distinguish between differences in cell cycle length and cell division timing.
Cell cycle kinetics, as well as the expression of p27Xic
(cdkn1c - ZFIN), did not significantly differ between the MIZ and LIZ
in any of the more than 50 embryos analyzed
(Fig. 5C, and data not shown).
These observations suggest that the cell division characteristics of the MIZ
and LIZ are comparable, and that these populations differ in their commitment
in a manner independent of cell cycle control.
Gli1 counteracts the IZ neurogenesis inhibition process and accounts for the differential sensitivity of MIZ and LIZ cells to neurogenesis inhibitors
Among commitment factors expressed within the early neural plate,
transcription factors of the Gli family have been implicated in neurogenesis
control in many systems (reviewed by
Agathocleous et al., 2007;
Ruiz et al., 2002).
Furthermore, Gli factors have been identified as intracellular targets for the
PKA/Gsk3β pair (Huangfu and Anderson,
2006; Riobo and Manning,
2007). gli1 appears to be expressed differentially
between the MIZ and LIZ: in agreement with published data
(Karlstrom et al., 2003), we
observed that gli1 is transcribed within the anterior neural plate
following a clear mediolaterally decreasing gradient starting at 80% epiboly
(Fig. 6A,C,E, and data not
shown). Double staining for GFP, identifying the IZ in
Tg(her5PAC:EGFP)ne1939 embryos, further confirmed that
this gradient is also found at the level of the IZ
(Fig. 6B,D,F). Three other Gli
genes have been identified in zebrafish
(Karlstrom et al., 2003;
Ke et al., 2005;
Tyurina et al., 2005).
gli2b is not expressed at detectable levels within the IZ area at
these stages, whereas expression of gli2a and gli3 is
ubiquitous (not shown).
To address the function of Gli1 during IZ formation, we blocked
gli1 translation by injection of a gli1 morpholino (MO)
(Karlstrom et al., 2003) into
wild-type embryos. This did not alter the IZ area (82% of cases,
n=71) (Fig. 6, compare
H,L with J,N; Fig. 7, compare E
with A), although it was efficient at blocking expression of the Hh target
nkx2.1 along the ventral midline of the anterior neural tube (96% of
cases, n=30) (not shown). However, blocking Gli1 totally abolished
the neurogenic effect of LiCl: in gli1 MO-injected embryos subjected
to LiCl treatment at 80% epiboly, the MIZ formed normally (82% of cases,
n=52) (Fig. 6, compare
I with G). Thus, blocking Gli1 function can increase neurogenesis inhibition
and rescue IZ formation in the absence of Gsk3β activity. Using a similar
approach, we tested whether blocking Gli1 could compensate for the lack of PKA
activity, by co-injecting gli1 MO and dnReg capped RNA into
wild-type embryos. The co-inhibition of Gli1 and PKA abolished the neurogenic
effect of blocking PKA alone and rescued MIZ formation (84% of cases,
n=61) (Fig. 6, compare
M with K). These results demonstrate that Gli1 exerts a neurogenesis-promoting
activity that opposes the activity of Gsk3β/PKA. Under normal conditions,
the activity of Gli1 is sub-threshold, such that neurogenesis inhibition is
obtained. It becomes visible in the absence of Gsk3β or PKA activity,
when the repression of Gli1 alone suffices to restore neurogenesis
inhibition.
|
The neurogenesis-promoting function of Gli1 and its MIZ-specific expression make it a good candidate to enhance the neurogenesis potential of the MIZ. As described, blocking Gli1 activity increases MIZ sensitivity to two copies (or fewer) of Her5/Her11 (Fig. 7F,G). In addition, in the absence of Gli1, we observed that both the MIZ and LIZ concomitantly lost their responsiveness to a further downregulation of Her5/Her11 to now upregulate neurog1 in an identical manner (90% of cases, n=56) (Fig. 7H,H'). These results identify Gli1 as a crucial element rendering MIZ and LIZ cells differentially sensitive to E(Spl) factors.
IZ formation and Gli1 activity are not under the control of Hh signaling
We next searched for mechanisms controlling Gli1 expression or activity
within the MIZ. Hh signaling is active in the presumptive axial mesoderm from
50% epiboly onwards (Ertzer et al.,
2007; Krauss et al.,
1993), and Gli1 behaves as a classical positive activator of Hh
targets. Thus, we studied whether IZ formation and Gli1 expression require Hh
signaling. We blocked Hh signaling upstream of PKA/Gsk3β action by
incubating wild-type embryos in cyclopamine. Cyclopamine blocks the
transmembrane protein Smoothened (Chen et
al., 2002), which normally initiates intracellular Hh signaling
events and in particular the inhibition of Gsk3β and PKA
(Huangfu and Anderson, 2006;
Riobo and Manning, 2007). As
expected, such treatment performed at 50% epiboly efficiently inhibited
expression of myoD in adaxial cells (97% of cases, n=40)
(Fig. 8, compare H with G)
(Barresi et al., 2000;
Chen et al., 2001) and of
spalt in the anterior neural plate (not shown). Surprisingly,
however, cyclopamine treatment did not affect gli1 expression in the
IZ area at any stage (100% of cases, n>20 for each stage)
(Fig. 8A-F, and data not
shown). Whatever the stage of application, cyclopamine also did not affect IZ
formation (Fig. 8, compare J-L
with N). Because Hh signaling might promote Gli1 activity rather than
expression, we assessed whether cyclopamine downregulated Gli1 function. For
this, we first reduced E(Spl) activity using her5GripNA, and next
applied cyclopamine at 50% epiboly. Whereas inhibiting Gli1 function in such
cases led to a rescue of the MIZ (Fig.
7F), cyclopamine was without effect and was incapable of
counteracting ectopic neurog1 induction (89% of cases, n=75)
(Fig. 8, compare M with I). We
conclude that cyclopamine treatment affects neither the expression nor the
function of Gli1 at the IZ. Similar results were obtained in
smub641 mutants, deficient in Smoothened function
(Varga et al., 2001), analyzed
for gli1 expression at 70% epiboly, tailbud and 3 somites
(n>30 embryos in each case) (not shown). Finally, we also observed
that gli1 expression and IZ formation were unaffected when
transduction of Hh signaling was blocked upon overexpression of a
Hh-insensitive form of the receptor Patched, Patchedloop2
(Briscoe et al., 2001) (100% of
cases, n>100) (Fig.
8, compare O with P), even in the absence of Her5 function (not
shown). These observations strongly suggest that the neurogenic activity of
Gli1 at the IZ does not require Hh signaling.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). We previously demonstrated that E(Spl) activity
is a crucial mechanism inhibiting neurogenesis to permit IZ formation
(Geling et al., 2003;
Geling et al., 2004;
Ninkovic et al., 2005). We
identify here a collaborative mechanism, relying on the activity of the two
kinases, PKA and Gsk3β. The E(Spl) and Gsk3β/PKA pathways act in a
dose-dependent manner, and one of their activities is to oppose the neurogenic
effect of the transcription factor Gli1. Gli1, expressed medially, enhances
the tendency of MIZ cells to undergo neurogenesis, a mechanism that might
account for the earlier differentiation of basal versus alar plate neurons
during MH development. Together, these results help refine a molecular model
for the sequential differentiation of midbrain-hindbrain neurons along the AP
and mediolateral (DV) axes (Fig.
9).
Co-regulation of IZ formation by the Gsk3β/PKA and E(Spl) pathways
An important finding of our work is the identification of Gsk3β and
PKA as new mediators of IZ formation. Both enzymes often act sequentially on
the same targets in vivo, PKA priming target proteins for a subsequent
phosphorylation by Gsk3β (Price and
Kalderon, 2002), but alternative models are emerging in which PKA
and Gsk3β independently phosphorylate the same target
(Taurin et al., 2006). At the
IZ, the enhanced effect of lowering both Gsk3β and PKA, and the fact that
constitutive PKA activation can compensate for reduced Gsk3β, suggest the
involvement of an unconventional dose-dependent process incorporating the
level of activity of both enzymes. PKA and Gsk3β might be rate-limiting
for the full phosphorylation necessary to functionally modify the same target.
Alternatively, PKA and Gsk3β might act on distinct molecular targets,
cooperating in parallel pathways during IZ formation. Our results do not allow
us to distinguish between these possibilities.
E(Spl) and Gsk3β/PKA act in an additive manner, and our observations
support parallel activities of E(Spl) and Gsk3β/PKA on a dose-dependent
process sensitive to the global level of these pathways. Gsk3β/PKA and
E(Spl) inhibition might converge onto common targets promoting neurogenesis
inhibition, or have parallel targets cooperating in the neurogenesis
inhibition process. The direct targets of E(Spl) activity during IZ formation
remain unknown, although it is known to downregulate expression of
neurog1, coe2 and p27Xic (cdkn1c - ZFIN)
(Geling et al., 2004). Many
targets inhibited by Gsk3β/PKA phosphorylation have been identified,
including factors controlling cell cycle (e.g. N-myc1; Mycn)
(Kenney et al., 2004;
Mill et al., 2005),
neurogenesis and cell differentiation (e.g. Neurogenin 2, XNeuroD, Xash1 and
Mash1) (Ma et al., 2008;
Moore et al., 2002), or both
(e.g. β-catenin or Gli proteins) (reviewed by
Frame and Cohen, 2001;
Huangfu and Anderson, 2006;
Riobo and Manning, 2007). None
of our observations suggests a major role for cell proliferation control in IZ
formation, corroborating previous findings that neurogenesis does not
systematically follow cell cycle exit at the early neural plate stage
(Geling et al., 2003;
Hardcastle and Papalopulu,
2000; Harris and Hartenstein,
1991). Rather, in this system, entry into the
neurog1-positive state follows information unlinked to cell cycle
characteristics and we favor commitment factors as the main targets for the
Gsk3β/PKA pathway.
|
) but not
on Neurog1 (our observations) and, in the IZ, Gsk3β/PKA affects
neurog1 transcription rather than its activity. We failed to identify
Gsk3β or PKA target sites on Her5 and Her11 (not shown), but an indirect
role of Gsk3β/PKA on the activity of these factors cannot be excluded.
Gli1 is an obvious candidate target, as Gsk3β/PKA classically regulate
the nuclear translocation (Gli1) or processing (Gli2, Gli3) of Gli factors in
a manner antagonized by Hh signaling
(Huangfu and Anderson, 2006;
Riobo and Manning, 2007).
However, Gsk3β/PKA also block neurogenesis in the LIZ, where
gli1 is not expressed. Hence, other direct targets of Gsk3β/PKA
remain to be identified that permit IZ formation.
Finally, as with E(Spl) factors, the upstream pathways involving
Gsk3β/PKA at the IZ are currently unknown. Classically, Wnt and Hh
inhibit Gsk3β and/or PKA to permit signal transduction (reviewed by
Hooper and Scott, 2005;
Huangfu and Anderson, 2006;
Logan and Nusse, 2004;
Riobo and Manning, 2007).
However, Wnt reporters remained silent in the anterior neural plate at the
stage of interest, and blocking Wnt or Hh activities or ubiquitously enhancing
PKA activity did not expand the IZ. IZ formation might be achieved through a
constitutive activation of Gsk3β/PKA in concert with local cofactors,
and/or through signaling pathways other than Wnt and Hh. A number of upstream
regulators have been identified for these kinases - in particular, classical
mitogenic pathways driven by PI3 kinase and Akt
(Dudek et al., 1997;
Kenney et al., 2004). It will
be important to test their influence during IZ formation.
Gli1 activity renders MIZ cells less sensitive to E(Spl)- and Gsk3β/PKA-mediated neurogenesis inhibition
gli1 expression is concentrated medially and has a
neurogenesis-promoting effect, albeit at sub-threshold levels. Significantly,
blocking Gli1 activity abolishes the difference between MIZ and LIZ
sensitivity towards E(Spl)-mediated neurogenesis inhibition. Although multiple
additional blocks might still exist between neurog1 expression and
the completion of the neuronal differentiation program, the mechanism
uncovered here is likely to facilitate the transition of MIZ cells towards
neuronal production and might be relevant to the earlier maturation of the
midbrain basal plate that is observed in all vertebrates during development
(Easter et al., 1994;
Puelles et al., 1987;
Ross et al., 1992;
Wilson et al., 1990). Although
E(Spl) and Gli1 activities have opposing effects on neurogenesis at the IZ,
our triple knockdown experiments of Gli1, Her5 and Her11 demonstrate that Gli1
itself is dispensable for neurog1 expression in the absence of the
inhibitory activity of E(Spl) factors. Gli1 activity might, however, be
required in this context for the progression of neuronal differentiation
beyond neurog1 expression, an issue that remains to be tested.
Gli1 exerts positive and negative effects on neurogenesis depending on the
cell type or state. Neurogenesis progression fails when Gli1 function is
blocked in the Xenopus neural plate or zebrafish retina
(Masai et al., 2005;
Nguyen et al., 2005). By
contrast, downregulation of Gli1 prevents re-entry into S phase in the chicken
ventral neural tube (Cayuso et al.,
2006). Overall, Gli1 might bring cells closer to differentiation,
hence, pushing early progenitors to amplify, and later progenitors to a final
cell cycle. At the IZ, however, where an amplifying population has not been
observed, an effect on the cell cycle is improbable and we propose that Gli1
targets cell cycle-independent commitment genes. Further arguments supporting
this hypothesis stem from previous analyses of zebrafish gli1
(detour) mutants (Karlstrom et
al., 1999). These mutants lack cranial motoneurons, including
nerve III (Chandrasekhar et al.,
1999) [which is likely to originate from the MIZ
(Tallafuss and Bally-Cuif,
2003) (K. Webb and C.S., unpublished)], but display no defects in
neurogenesis at 18 hours, pointing to a differentiation rather than an
induction defect (Chandrasekhar et al.,
1999). However, the lack of cranial motoneurons of gli1
mutants can be mimicked by loss of Smoothened function or by cyclopamine
treatment (Vanderlaan et al.,
2005), and hence might be a result of impaired Hh signaling, which
contrasts with our interpretation of Gli1 regulation at the MIZ. In any case,
both analyses point to a role for Gli1 in promoting differentiation in a
manner independent of cell cycle control.
Hh-independent regulation of Gli1
Given the importance of Gli1 function in neurogenesis progression, it
remains crucial to uncover the pathway(s) controlling Gli1 expression in the
MIZ. Gsk3β/PKA can downregulate Gli1
(Huangfu and Anderson, 2006;
Riobo and Manning, 2007) and
might do so during IZ formation. This is likely to occur at the
post-transcriptional level, as gli1 transcript levels were not
modified by Gsk3β or PKA blockade (not shown). However, this
downregulation of Gli1 by Gsk3β/PKA is only partial, because blocking
Gli1 activity has further phenotypic consequences.
A most surprising aspect of our study is that Gli1 expression and activity
at the IZ are not under the control of Hh signaling alone. Blocking Hh with
cyclopamine, in smoothened (smu) mutants or by
overexpressing a Hh-insensitive form of Patched, affected neither
gli1 expression nor IZ formation. Although surprising, these findings
are in keeping with previous observations by Karlstrom et al. who reported
maintenance of gli1 expression in the anterior neural plate of
smu mutants or cyclopamine-treated embryos
(Karlstrom et al., 2003).
Recently, a discrepancy between the effects of smu mutations or
cyclopamine and the effects of forskolin, which activates PKA, were noted
during retinal neurogenesis in zebrafish: whereas forskolin strongly impaired
neurogenesis progression, smu mutant or cyclopamine-treated embryos
exhibited only mild defects (Masai et al.,
2005). Our results are comparable to these observations and, we
believe, question the role of Hh, if any, in Gli1 regulation across the IZ. In
a few instances, activation of Gli1 (or Gli2) has been reported to follow
activation of the Erk pathway by Fgf signaling
(Brewster et al., 2000;
Riobo et al., 2006a;
Riobo et al., 2006b) or of the
PI3K and Akt pathways by mitogens (Riobo
et al., 2006b). It is possible that such an atypical,
Hh-independent mechanism is involved at the midline of the anterior neural
plate. Unravelling this process will be important to our understanding of
neurogenesis control and the cross-regulatory activities of Hh signaling
pathway components.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Present address: Helmholtz Zentrum Muenchen, German Research Center for
Environmental Health, Institute of Stem Cell Research, Ingolstaedter
Landstrasse 1, D-85764 Neuherberg, Germany
|
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