Department of Neuroscience and Cell Biology, UMDNJ/Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA
* Author for correspondence (e-mail: matisemp{at}umdnj.edu)
Accepted 22 April 2004
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SUMMARY |
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Key words: Sonic hedgehog, Gli genes, Patterning, Cell fate, Spinal cord, Sorting, Mouse, Chick
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Introduction |
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The Ci gene encodes a zinc-finger-containing transcription factor
with two distinct activities, repression and activation
(Aza-Blanc and Kornberg, 1999).
Hh signaling controls these dual activities by promoting the formation of a
full-length Ci activator protein at the expense of the shorter repressor form
that is constitutively generated by partial proteolysis in the absence of Hh
(Aza-Blanc et al., 1997
). The
differential sensitivity of Hh target genes to the two forms of Ci indicates
that activation of Hh target genes involves both disinhibition and direct
activation (Muller and Basler,
2000
). Similarly, evidence suggests that vertebrate Gli2 and Gli3,
but not Gli1, proteins can be cleaved to generate repressor forms, analogous
to Ci (Dai et al., 1999
;
Wang et al., 2000
;
Aza-Blanc et al., 2000
).
However, the dual transcriptional activities embodied in Ci appear to be
unequally distributed among the three vertebrate Gli proteins. Both gain- and
loss-of-function studies indicate that Gli1 and Gli2 are the primary Gli
activators that function downstream of Shh signaling, but only Gli2 is
required in developing mice (Ruiz i
Altaba, 1999
; Ding et al.,
1998
; Matise et al.,
1998
; Bai et al.,
2002
). Gli3, however, appears to function as the primary Gli
repressor (Wang et al., 2000
),
although biochemical and genetic evidence suggests that Gli3 can also function
as an activator under certain circumstances
(Dai et al., 1999
;
Shin et al., 1999
;
Motoyama et al., 2003
;
Bai et al., 2004
). A repressor
role for Gli2 has not been demonstrated.
All three Gli genes are expressed in spinal cord progenitor cells during
early neurogenesis when cell fate specification is occurring
(Sasaki et al., 1997;
Platt et al., 1997
;
Lee et al., 1997
). However,
mouse mutant studies reveal only a limited role for each individual Gli gene
in dorsoventral (DV) patterning in the spinal cord. Targeted Gli2
mutant mice lack floor plate (FP) and most V3 cells that develop near the
ventral midline, but other ventral cell classes are present in their normal DV
positions, except motoneurons (MNs), which extend across the midline
(Ding et al., 1998
;
Matise et al., 1998
).
Gli1 mutants have no discernable spinal cord phenotype, even on a
Gli2 mutant background (Matise et
al., 1998
; Park et al.,
2000
), while loss of Gli3 has only a subtle effect on the
position of interneurons that develop in the intermediate region of the spinal
cord (Persson et al.,
2002
).
By contrast, Shh mutants have a severe phenotype including
cyclopia and an absence of most ventral cell types along the entire neuraxis
(Chiang et al., 1996). In
Shh;Gli3 and Smo;Gli3 double mutants, many ventral cells are
rescued except FP and V3 interneurons
(Litingtung and Chiang, 2000
;
Wijgerde et al., 2002
),
indicating that an important function of Hh signaling is to oppose the
repressive activities of Gli3 in the ventral spinal cord. In addition, these
results, and others (Krishnan et al.,
1997
), suggest the possibility that a Hh/Gli-independent pathway
could mediate some aspects of Shh signaling in the ventral spinal cord.
However, as Gli2 is still expressed in Shh;Gli3 and Smo;Gli3
mutants, a Hh-independent role for this factor in generating some ventral cell
types cannot be ruled out.
Together, these studies suggest a model whereby graded Shh signaling
controls the balance between Gli activator and repressor activities in
progenitor cells along the DV axis, and predicts that the summation of Gli
activities at specific Shh concentrations (and DV levels) will control
distinct cell fates, ultimately by regulating expression of progenitor fate
determinants (Stone and Rosenthal,
2000; Jacob and Briscoe,
2003
). However, a number of issues remain unresolved. First, it is
unclear which specific cell fates and progenitor determinants are controlled
by the different Gli activities. For example, while it has recently been
demonstrated that many ventral class II determinants are sensitive to Gli3
repression (Persson et al.,
2002
; Meyer and Roelink,
2003
), whether and how specific Gli activator activities are
involved in controlling the expression of these factors in spinal cord DV
patterning has not been resolved. Second, it is unclear whether Gli3 plays a
positive role in mediating Hh signaling. Indeed, it has recently been shown
that loss of Gli2 or Gli3, but not Gli1, can
reverse aspects of the Ptch1-/- mutant phenotype in which
the Hh pathway is constitutively activated
(Bai et al., 2002
;
Motoyama et al., 2003
),
suggesting that they both possess the ability to transduce Hh signaling as
activators in vivo. Finally, it is uncertain whether the Hh-dependent
establishment of neuronal patterning and ventral cell fates requires all Gli
protein functions. Resolving this issue is the rate-limiting step in
determining the contribution of Hh-independent pathways to ventral cell fate
specification and patterning.
In this study, we have addressed these issues using two approaches. First,
we generated mouse embryos lacking both Gli2 and Gli3 genes
and analyzed spinal cord development during early neurogenesis. As
Gli1 is not expressed in double homozygotes, all Gli protein
activities are absent in these mice, providing an opportunity to address the
requirement for all three vertebrate Gli factors and their combined activities
in mediating Hh signaling in the developing spinal cord. To complement these
studies, we employed gain-of-function experiments in chick embryos to study
the individual transcriptional activities of Gli2 and Gli3. Our results show
that motoneurons and three ventral interneuron subclasses are generated in
Gli2;Gli3 mutants, except floorplate and V3 cells, but strikingly
these cells develop as intermingled populations. Furthermore, we show that
Gli3 contributes activator functions to ventral neuronal patterning, playing a
redundant role with Gli2. The similarities of these results to previously
published studies (Litingtung and Chiang,
2000; Wijgerde et al.,
2002
), indicate that Gli proteins mediate all of the patterning
functions of Hh in the developing spinal cord. We also show that activation of
the Shh pathway in chick dorsal neural tube cells using Gli2 and Gli3
activator constructs, as well as expression of Shh, elicits cell clustering.
Together, these results indicate both distinct and partially overlapping roles
for Gli2 and Gli3 activator in patterning and cell fate specification in the
ventral spinal cord downstream of Shh signaling, and suggest an important role
for these activities in establishing or maintaining the segregation of ventral
progenitors in discrete pools.
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Materials and methods |
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In ovo electroporation in chicken embryos
cDNAs encoding amino acids 280-1544 of mouse Gli2 and 345-1596 of mouse
Gli3 were cloned into the bi-cistronic expression vector pCIG
(Sasaki et al., 1999;
Megason and McMahon, 2002
). An
ATG codon was added in frame upstream of both constructs, and was confirmed by
sequencing. For co-electroporation of these constructs with ptc
loop2,
the GFP was removed and ptc
loop2 cDNA
(Briscoe et al., 2001
) was
subcloned into pCIG. Five 25 V pulses were delivered for 50 mseconds at 1
second intervals at HH stages 12-14. Embryos were sacrificed 24 or 48 hours
later and processed for analysis. At least 10 embryos were analyzed for each
set of experiments. Noon on the day when vaginal plugs were detected was
designated E0.5.
Immunohistochemistry, RNA in-situ hybridization and RTPCR
Embryo collection, antibody staining and RNA in situ hybridization was
performed as described (Matise et al.,
1998). Antibodies used were mouse anti-BrdU (Sigma), cyclin D1
(Upstate Biotechnology), Shh, Nkx2.2, Hb9/Mnr2, Isl1, Pax6, Pax7, Foxa2
(DSHB), Gata3, Jag1 (Santa Cruz), Mash1 and Ngn1 (D. Anderson); rabbit
anti-phosphorylated caspase 3 (Idun Pharmaceuticals), Dbx1, Dbx2, Nkx6.1 (T.
Jessell), Chx10 (K. Sharma) and Olig2 (H. Takebayashi); and guinea-pig
anti-Nkx2.9, Nkx6.2 (J. Ericson) and Evx1 (T. Jessell).
Fluorochrome-conjugated secondary antibodies were obtained from Jackson
ImmunoResearch or Molecular Probes. RNA in situ probes were mouse
Ptch1 (M. Scott), chicken Ptch1 and Ptch2 (C.
Tabin). RT-PCR was performed as described using primers specific for mouse
Gli1 (Park et al.,
2000
).
Quantification of neurons and precursors was performed by averaging counts from wild-type and double-homozygous embryos. At least four sections for each set of markers were counted in three to eight different embryos at similar anterior-posterior levels.
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Results |
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In wild-type embryos, Shh expression is first seen in notochord precursors
and then later in prospective floorplate (FP) cells in the midline of the
neural plate. At E10.5, after neural tube closure, Shh expression is seen in
both the FP and notochord (Fig.
1C,D). In Gli2-/- mutant embryos that lack a
FP, Shh is detected only in the notochord
(Fig. 1E,F)
(Ding et al., 1998;
Matise et al., 1998
), while
Gli3xt/xt mutants are indistinguishable from wild type
(Fig. 1G,H). In
Gli2-/-;Gli3xt/xt mutants, Shh was
detected in the notochord at all axial levels examined, but as in
Gli2 mutants the FP did not form and no Shh expression was seen in
the ventral midline (Fig.
1I,J).
Ptch1 expression is a reliable indicator of Shh signaling as its
transcription is de-repressed by Shh
(Goodrich and Scott, 1998). In
wild-type embryos, Ptch1 expression is strong in VZ cells near the
ventral midline and weaker in the ventral VZ, but is undetectable in the
dorsal VZ (Fig. 1K). In
Gli2-/- mutants, weak Ptch1 expression was seen
in the ventral VZ but the strong expression adjacent to the FP was not
detected, probably owing to the absence of most V3 cells in these embryos
(Fig. 1L). By contrast,
Gli3xt/xt mutants showed strong ventral expression
adjacent to the FP as in wild type, but weak ectopic expression was also
detected in the dorsal VZ (Fig.
1M), suggesting that Gli3 normally represses
Ptch1 transcription here. Interestingly, in
Gli2-/-;Gli3xt/xt double mutants, low
levels of uniform Ptch1 expression were detected in a pattern that is
essentially a composite of the Gli2 and Gli3 single mutant
patterns (Fig. 1N). By
contrast, Ptch2, which is expressed only at low levels in the neural
tube (Motoyama et al., 1998
),
was not affected in double mutants (data not shown). These results show that
Ptch1 can be transcribed at basal levels in the absence of Gli
activity but requires both Gli2 and Gli3 to establish its normal pattern of
expression in the spinal cord.
We noted overgrowth of the neuroepithelium in the thoracic regions of some Gli2-/-;Gli3xt/xt mutants, suggesting a proliferative defect (n=3/11 embryos at E10.5; see Fig. S1 at http://dev.biologists.org/supplemental; see also Fig. 1N, Fig. 3R) This was accompanied by an increase in cell cycle markers in affected areas, but was not confined specifically to dorsal or ventral regions (see Fig. S1 at http://dev.biologists.org/supplemental). The majority of Gli2-/-;Gli3xt/xt mutants did not exhibit this phenotype, and cell proliferation was similar to wild type (see Fig. S1 at http://dev.biologists.org/supplemental). This finding indicates that loss of both Gli2 and Gli3 lead to sporadic defects in cell proliferation independent of patterning defects, which were confined to the ventral spinal cord.
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Three interneuron subclasses develop in the ventral spinal cord dorsal to
MNs and can be identified by their expression of Chx10 (V2), En1 (V1) and Evx1
(V0). V2 cells can be further subdivided into two subclasses, V2a (Chx10+) and
V2b (Gata3+), that develop as intermingled cells
(Karunaratne et al., 2002)
(Fig. 3D). In
Gli2-/-;Gli3xt/xt mutants, V1, V2a and
V2b cells were present but were not confined to their normal domains dorsal to
MNs (Fig. 3B,E,H). Rather,
these cells expanded into the ventral midline where they were intermingled
with one another and MNs in both thoracic
(Fig. 3H,K) and lumbar (data
not shown) regions. V0 neurons, by contrast, remained largely in their normal
domain, although some mixing with V1 cells was seen at their dorsal boundary
(Fig. 3H). Cell counts revealed
a significant increase in V0 and V1 cells in thoracic, but not lumbar, regions
at both E10.5 (Fig. 3I) and
11.5 (data not shown). By contrast, fewer V2 cells were found in both regions,
similar to MNs (Fig. 3F). In no
case did we observe inappropriate co-expression of neuronal markers in double
mutant embryos at E10-10.5. Thus, in the absence of Gli activity, MN, V2 and
V1 interneurons are generated as intermingled, rather than distinct, cell
populations.
Abnormal MN differentiation and intermingling of MN, V1 and V2 progenitors in Gli2-/-;Gli3xt/xt mutants
To study the reduction in MN numbers in double mutant embryos, we examined
the expression of MN progenitor (pMN) factors. In
Gli2-/-;Gli3xt/xt mutants, Olig2+ pMN
cells were shifted ventrally in both thoracic and lumbar regions
(Fig. 3L-P). Interestingly, in
thoracic regions a higher numbers of pMN cells were seen in
Gli2-/-;Gli3xt/xt mutants compared
with wild type (20% increase), while in lumbar regions there were fewer
(
15% decrease) (Fig. 3N;
n=21 sections scored in three embryos; data not shown). Thus, the
reduction in postmitotic MNs generated in
Gli2-/-;Gli3xt/xt mutants is not due
solely to depletion of pMN cells. In addition, this decrease was not
correlated with upregulation of cyclin D1 or downregulation of Ngn1 expression
in pMN cells, nor increased MN apoptosis in
Gli2-/-;Gli3xt/xt mutants (see Fig. S1
at
http://dev.biologists.org/supplemental).
These findings suggest that the large reduction in the number of MNs in
Gli2-/-;Gli3xt/xt mutants in both
thoracic and lumbar regions could be due to a delay or decrease in the
progression of motoneuron progenitors to post-mitotic neurons.
We next examined Pax6, which shows weak expression in pMN cells and strong
levels in p2, p1 and p0 progenitor cells
(Fig. 3Q)
(Ericson et al., 1997). In
Gli2 mutants, Pax6 is repressed in the ventral pMN domain, a finding
that could be explained by reduced but persistent Nkx2.2/Nkx2.9 expression in
these mice (Fig. 2C,D)
(Ding et al., 1998
;
Matise et al., 1998
).
Consistent with this, in
Gli2-/-;Gli3xt/xt mutant embryos, both
Nkx2.2 and Nkx2.9 are completely absent and Pax6 expression extended into the
ventral midline at E10.5 and E11.5 (Fig.
3R; data not shown). By contrast, Pax7 expression, which marks
dorsal progenitors, was not altered in
Gli2-/-;Gli3xt/xt mutants (data not
shown).
In the ventral spinal cord, the five cardinal neuronal subtypes derive from
progenitor domains (ventrally to dorsally: p3, pMN, p2, p1 and p0) in which
cells express unique HD and basic helix-loop-helix (bHLH) proteins
(Briscoe and Ericson, 2001).
pMN and p2 cells can be identified by their expression of Olig2 and Mash1,
respectively, while both progenitor domains express Nkx6.1
(Fig. 4A)
(Lu et al., 2002
;
Zhou and Anderson, 2002
;
Parras et al., 2002
). In
Gli2-/-;Gli3xt/xt mutants,
Nkx6.1+/Mash1+ p2 progenitors were scattered throughout the ventral spinal
cord (Fig. 4B). Dbx2, which
normally defines the p1, p0 and pd6 progenitor domains
(Pierani et al., 1999
), was
also detected in scattered cells throughout the ventral spinal cord in
Gli2-/-;Gli3xt/xt mutants
(Fig. 4C,D). By contrast, Dbx1
expression, which marks p0 cells (Pierani
et al., 1999
), was confined to its normal intermediate domain,
consistent with the localization of V0 interneurons to this region in
Gli2-/-;Gli3xt/xt mutants (data not
shown).
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Olig2 expression in pMN cells is flanked by Mash1 expression in p3 and p2 cells (Fig. 5A). In Gli2-/- mutants, the ventral domain of Mash1 is absent, but the pMN and p2 domains segregate normally (Fig. 5B). By contrast, in Gli2-/-;Gli3xt/xt double mutants, Mash1+ (p2) and Olig2+ (pMN) cells were intermingled throughout the ventral region, but no cells were found that co-express these factors (Fig. 5C). Thus, pMN and p2 progenitors are intermingled but distinct in Gli2-/-;Gli3xt/xt mutants.
|
These data show that in the absence of Gli activities, pMN, p2 and p1 progenitors are generated in the ventral spinal cord and maintain their distinct identities, despite developing as intermingled populations (summarized in Fig. 5G-I).
Dominant Gli2 and Gli3 activator proteins induce ventral and repress dorsal cell fates
Our analysis of Gli2-/-;Gli3xt/xt
mutants shows that Gli2 and Gli3 play redundant roles in the ventral spinal
cord. However, loss-of-function studies do not permit us to address which
specific Gli activity - activation or repression - is involved, because all
are absent in double mutants.
To investigate this, we assayed the expression of ventral HD and bHLH fate
determinants in the spinal cord of chick embryos after transfection, shortly
after neural tube closure, of dominant constitutive Gli2 and Gli3 activator
constructs. To generate these, we deleted the region encoding the N-terminal
repressor domains of Gli2 and Gli3 (Sasaki
et al., 1999; Pearse et al.,
1999
; Murone et al.,
2000
; Dunaeva et al.,
2003
) to generate Gli2
N-term and Gli3
N-term (see
schematic, Fig. 6), and cloned
these constructs into a bi-cistronic expression vector
(Megason and McMahon, 2002
)
that also encoded GFP.
|
We found that Gli2N-term transfections induced cell-autonomous
expression of multiple ventral markers, including Foxa2 (FP,V3), Nkx2.2 (V3),
and (weakly) Nkx 6.1 (p3, pMN, p2) (Fig.
6C,E,G). By contrast, Gli3
N-term only induced Nkx 2.2 in
these experiments (Fig. 6F). No
effect was seen on Ngn2 or Nkx6.2 expression for either construct (data not
shown). Both Gli2
N-term and Gli3
N-term were equally capable of
repressing Pax6 and Pax 7 expression (Fig.
6I-L'). These results show that dominant Gli2 and Gli3
activator constructs are capable of inducing expression of a limited set of
ventral Class II fate determinants, with Gli2
N-term showing a broader
capability compared with Gli3
N-term, whose activity appears to be
confined primarily to the induction of Nkx2.2.
Interestingly, 1/3 of Gli2
N-term transfected embryos also
exhibited an expansion of the neuroepithelium on the transfected side
(n=7/20 embryos), which resulted in a unilateral increase in the
expression of cell cycle markers (Fig.
6A',C',E'; data not shown). Gli3
N-term
transfections, however, did not alter the size or shape of the
neuroepithelium.
We next assayed whether Gli2N-term and Gli3
N-term activator
constructs could influence the transcription of Ptch1 and
Ptch2. Mis-expression of both factors upregulated strong
Ptch2 expression (Fig.
6N,P), while Gli2
N-term was more efficient than
Gli3
N-term in inducing Ptch1
(Fig. 6M,O). These results
suggest that Ptch1 and Ptch2 are differentially responsive
to Gli2 and Gli3 activators.
To rule out the possibility that Gli2N-term or Gli3
N-term
induced the expression of Shh at low levels that escaped detection, we
co-transfected these constructs with ptc-
loop2, a dominant inhibitor of
Shh signaling (Briscoe et al.,
2001
). On its own, ptc-
loop2 can block Nkx 2.2 and activate
Pax7 expression (see Fig. S2 at
http://dev.biologists.org/supplemental)
(Briscoe et al., 2001
).
Co-transfection of Gli2
N-term or Gli3
N-term with
ptc-
loop2 overcame these effects and instead activated Nkx2.2 and
repressed Pax7 expression, and for all markers examined co-transfection
results were similar to Gli2
N-term or Gli3
N-term alone (see Fig.
S2 at
http://dev.biologists.org/supplemental;
data not shown). These results show that alterations in gene expression after
transfections with Gli2
N-term and Gli3
N-term activator
constructs cannot be explained by the secondary induction of Shh, but instead
are the result of these factors acting in a ligand-independent cell-autonomous
manner to induce target gene expression.
Activation of the Shh pathway in dorsal cells elicits cell clustering
The non-uniform expression of induced Ptch1 and Ptch2 by
Gli2 and Gli3 activators, and the expansion of the neuroepithelium elicited by
Gli2 activator transfections indicate that these factors might possess
activities that are not directly related to control of progenitor fate
determinant gene expression. To study this, we examined the behavior of
transfected cells within the Pax7 domain in the dorsal spinal cord. In these
experiments, we were guided by similar studies in Drosophila wing
imaginal discs which showed that manipulation of the Hh pathway in anterior
and posterior compartment cells could modify cell affinities between Hh
transducing and non-transducing cells
(Dahmann and Basler, 2000). In
addition, recent experiments in mice provided evidence for a similar function
of the Hh pathway in spinal cord patterning. In this study, Hh signaling was
blocked in ventral cells by generating chimeras with
Smo-/- ES cells, and it was found that these cells
`clumped' together in the ventral spinal cord
(Wijgerde et al., 2002
). We
reasoned that if the Shh pathway functioned in part to segregate ventral from
dorsal cells, then activating the pathway in dorsal cells should also elicit
similar clustering behaviors.
We first transfected Shh alone into the spinal cord and monitored Nkx2.2 and Pax7 expression to identify cells in which the pathway has or has not been activated, respectively. Not unexpectedly, ectopic Shh induced widespread, almost uniform expression of Nkx2.2, and repressed Pax7 in both cell-autonomous and non-autonomous manners (Fig. 7A,B,D). In this experiment, Nkx2.2-expressing cells did not segregate from one another or from dorsal cells. As a control for these experiments, we transfected GFP alone, which did not elicit cell clustering or alterations in either Nkx2.2 or Pax7 expression (Fig. 7C).
|
These results show that activation of the Shh pathway in dorsal cells can
elicit cell clustering. To determine whether Gli activators mediated this
effect, we transfected Gli2N-term or Gli3
N-term constructs and
examined transfected embryos that exhibited broad transfections extending into
the dorsal spinal cord. In these experiments, we found that in a majority of
sections examined (n=13/17 sections, 10 embryos), transfected cells
that did not show Pax7 expression were found in clusters, surrounded by
non-transfected Pax7+ cells (Fig.
7I-L), although ventrally located transfected cells were
distributed more uniformly (n=14 sections; data not shown;
Fig. 7M). Similar results were
obtained at the lowest Gli2
N-term concentration that induced Nkx2.2
expression (n=3; data not shown). Thus, cell clustering elicited by
activation of the Shh pathway in dorsal neural tube cells appears to be
mediated by Gli2 and/or Gli3 activator activities
(Fig. 7M,N).
To determine whether cell clustering that results from activation of the Shh pathway is mediated by induction of Nkx2.2, we transfected this factor alone and assayed the position of transfected cells in the dorsal spinal cord. In contrast to Shh or Gli2/Gli3 activator transfections, Nkx2.2-transfected cells did not segregate from non-transfected cells in the dorsal spinal cord in any case examined (n=8 sections, three embryos; data not shown). These results show that cell clustering induced by Shh-pathway activation is independent of Nkx2.2 induction in chick neural progenitors.
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Discussion |
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Unique and redundant roles for Gli2 and Gli3 activators in patterning ventral cells
Our current analysis of neuronal specification in Gli2 single and
Gli2;Gli3 double mutants indicates that Gli3 is partially redundant
with Gli2 for the expression of Gli1 and the generation of V3 cells.
In Gli2 mutants, Gli1 expression is severely reduced and the
FP and most V3 cells are absent (Matise et
al., 1998), whereas in Gli2;Gli3 mutants Gli1
expression and V3 cells are completely absent. Our transfection studies,
furthermore, show that dominant activator forms of both Gli2 and Gli3 are
capable of inducing ectopic expression of the V3 marker Nkx2.2 throughout the
neural tube. Together with previous studies
(Briscoe et al., 1999
;
Aza-Blanc et al., 2000
;
Bai and Joyner, 2001
;
Bai et al., 2004
), these
results demonstrate that Gli2 and Gli3 activators function redundantly to
define the p3 progenitor domain and specify V3 interneurons by controlling
expression of Nkx2.2 and perhaps Gli1.
Both Gli2 and Gli3 contain an N-terminal repressor domain that physically
interacts with Su(Fu), a negative regulator of the Shh pathway
(Pearse et al., 1999;
Murone et al., 2000
;
Dunaeva et al., 2003
).
Previous studies in transgenic mice showed that misexpression in the dorsal
midbrain of Gli2 and Gli3 proteins lacking this N-terminal domain, but
retaining the DNA-binding zinc finger and C-terminal activation domains
(Yoon et al., 1998
), could
activate ectopic expression of Foxa2/Hnf3ß
(Sasaki et al., 1999
).
Interestingly, we found that misexpression of N-terminally-deleted Gli2 and
Gli3 activators in chick spinal cords using in ovo electroporation elicited
different outcomes. Gli2 activated expression of Foxa2 (Hnf3ß), Nkx2.2
and Nkx6.1, as well as inducing an expansion of the neuroepithelium, while
Gli3 was only capable of inducing Nkx2.2. The striking similarity of these
activities to the differential requirements for Gli2 and Gli3 in specifying
ventral cell fates, as revealed by our analysis of Gli2;Gli3 double
mutants, suggests that these might reflect functional differences in the
capacity of Gli2 and Gli3 to activate Shh target genes. However, as
N-terminally-truncated forms of Gli2 and Gli3 are not detected in situ, we
cannot rule out the possibility that the different responses to
Gli2
N-term and Gli3
N-term in our transfection experiments do not
reflect true differences in the activities of these proteins in vivo.
Nevertheless, these results suggest that intrinsic functional differences
between Gli2 and Gli3 could play a role in differentiating the cellular
responses to Shh.
We found that Ptch1 and Ptch2 are differentially
responsive to constitutive Gli2 and Gli3 activators. Taken together with
previous studies showing that a Gli3 repressor can downregulate Ptch1
and Ptch2 transcription in the neural tube
(Persson et al., 2002), it is
likely that Ptch1 and Ptch2 are regulated by both Gli
activator and repressor activities in the developing spinal cord. In this
regard, the Ptch genes appear analogous to dpp in flies, the
expression of which is controlled by both activator and repressor forms of Ci
(Muller and Basler, 2000
).
Obligatory role for Gli proteins in mediating Hh-dependent neural patterning in the ventral spinal cord
Studies in Drosophila have shown all of the known responses to Hh
signaling require the activities of Ci
(Methot and Basler, 2001). In
Smo;Gli3 mutants, which are in theory incapable of responding to all
Hh proteins, many ventral cell types that are lost in Shh mutants are
rescued, but some populations are mixed in the ventral spinal cord
(Wijgerde et al., 2002
).
However, as Gli2 is still present in these mice as its transcription
is independent of Shh signaling (Bai et
al., 2002
), it was not possible to conclude that all Gli protein
activities are required for this potential function. Results from the present
study reveal numerous phenotypic similarities between Smo;Gli3 and
Gli2;Gli3 mutants, and provide support for the idea that Gli protein
activities are required for all the known patterning functions of Hh signaling
in the developing ventral spinal cord.
Role of Gli proteins in mediating graded Shh signaling in the developing spinal cord
From our studies and those from other laboratories, a clear picture has
emerged of the individual roles of Gli protein activities in mediating
cellular responses to graded Hh signaling in the developing spinal cord. All
three Gli factors contribute positive functions to the transduction of the Shh
signal in the ventral spinal cord, with Gli2 and Gli3 playing the predominant
roles to transduce the initial Shh signal and Gli1, which is induced by Gli2
and Gli3, making a minor contribution. Thus, all three Gli proteins
participate as activators in the induction of FP and V3 interneurons, while
Gli2 and Gli3 activators are also required for normal motoneuron
differentiation and V2-V0 interneuron patterning and number, but not
specification. Gli3 serves as the primary Gli repressor that must be inhibited
to allow normal ventral cell fate development, but this activity also appears
to be required for normal V2-V0 interneuron development independent of Hh
signaling (Persson et al.,
2002).
Available evidence does not, however, definitively exclude the possibility
that a Gli2 repressor could play a minor Hh-independent role in the spinal
cord. The increase in V1 interneurons in thoracic regions of
Gli2;Gli3 double mutants compared with normal embryos could be due to
the removal of a repressive function for Gli2 that normally serves to limit
the number of these cells. Interestingly, the situation in Smo;Gli3
embryos (that might retain Hh-independent Gli2 repressor activity) appears to
be the converse of this, with slightly more V1 cells in lumbar, but not
thoracic, regions (Wijgerde et al.,
2002). These findings suggest that if Gli2 does function as a
repressor in vivo, this activity is redundant with Gli3 and its role differs
along the rostrocaudal axis of the spinal cord. However, no direct evidence
has been provided to date demonstrating the presence of an active, truncated
repressor form of Gli2 in the developing spinal cord. Alternatively, as it has
been shown that V1 and V0 cells can be induced by retinoids
(Pierani et al., 1999
), it is
also possible that the normal formation of these cell types involves a correct
balance or integration of both Shh and retinoid signaling pathways, and that
this balance is differently affected in mouse Gli2;Gli3 and
Smo;Gli3 mutant backgrounds.
Our results also reveal a redundant role for Gli protein activities in normal motoneuron differentiation. We found that in mice lacking all Gli activities, postmitotic motoneurons were present in greatly reduced numbers relative to their precursors, which were either near normal in number or elevated (in thoracic regions). This defect is not explained by inappropriate up-regulation of cyclin D1 or downregulation of Ngn2 in motoneuron precursors (see Fig. S1 at http://dev.biologists.org/supplemental; Q.L and M.P.M., unpublished). One possibility is that in the absence of regulated Gli protein activities, motoneuron progenitors do not respond normally to differentiation cues that are transmitted to cells during or shortly after their terminal mitosis.
Gli2 and Gli3 activator activities contribute to Shh/Gli control of neuronal patterning by regulating progenitor segregation
Our analysis of neuronal progenitor markers in Gli2;Gli3 mutant
spinal cord reveals that p1 and p2 cells that develop in the ventral spinal
cord are mixed with each other and pMN progenitors. We show that these
progenitors maintain distinct identities despite being mislocalized in the
ventral spinal cord. As a result, neural patterning is similarly disrupted,
with V2, V1 and MNs forming as intermingled, rather than discrete,
populations. Within the context of current models, the simplest interpretation
of these results is that, in the absence of Gli activities, many ventral
precursors are incapable of responding normally to graded Shh signaling,
resulting in the random specification of progenitors and the generation of
similarly intermingled neuronal progeny. However, another interesting
explanation could also account for these results. Lineage tracing studies
indicate extensive intermixing of spinal cord progenitors prior to
neurogenesis (Erskine et al.,
1998). It is therefore conceivable that the mechanisms involved in
specifying positional identity in the spinal cord function in part by
restricting this intermixing. This function has been proposed for HD
progenitor fate determinants, the expression of which is initiated at the
onset of neurogenesis and which function to refine domains by mutual
cross-repression (Briscoe et al.,
2000
). However, with the exception of Nkx2.2, our results show
that Gli-mediated Shh signaling is not required for the induction or
cross-repressive functions of most ventral HD determinants but rather is
needed to organize their normal expression into distinct DV domains.
Furthermore, our gain-of-function studies in chick embryos suggest a role for the Shh/Gli signaling in establishing progenitor pool segregation in the spinal cord that is independent of their regulation of HD determinants. In these experiments, we activated the Shh pathway in dorsal cells using overexpression of ligand or N-terminally truncated Gli2/Gli3 activator proteins. In both cases, cells in which the pathway was activated were segregated from non-transducing dorsal cells when assayed 24 hours after transfection. As cell clustering cannot be induced by Nkx2.2 (the only HD factor consistently induced in these assays), it appears that distinct Gli target genes mediate this effect.
A role for Hh signaling in controlling progenitor segregation has also been
proposed based on recent studies in Smo-/- mouse mutants
(Wijgerde et al., 2002). In
this study, Hh signaling was blocked in ventral cells by generating chimeras
with Smo-/- ES cells, and it was found that progenitors
derived from these cells `clumped' together in the ventral spinal cord.
However, as Smo-mutant cells in chimeras are present throughout
embryogenesis, it is unclear whether Smo (or Hh signaling) is required in
these cells during neurogenesis (when cell fate specification is occurring) or
at some earlier time point. In addition, any Gli proteins present within
Smo-/- mutant cells are likely to be repressors, so
whether Gli activators, or Gli proteins in general, also participate in the
hypothesized role of Hh signaling in progenitor segregation was not addressed.
Our results establish that the opposite manipulation (activating the pathway
in dorsal cells) has a similar outcome. Together, these results demonstrate
that altering Shh pathway status in cells relative to their neighbors, either
positively (our study) or negatively
(Wijgerde et al., 2002
), can
induce segregation. Furthermore, cell segregation defects are only seen in
mouse mutants in which all Gli activator activities are absent
(Smo-/- chimeras, Smo;Gli3 and Gli2;Gli3
mutants). Together, these results illustrate a crucial role for Gli
activators, either on their own or in balance with Gli repressors, in
controlling progenitor segregation in the neural tube.
It is not immediately obvious from our studies how Gli activator-induced
cell clustering in the dorsal spinal cord is related to their normal role
ventrally. Pathway-activated cells in the ventral spinal cord did not form
clusters, even at 20-fold lower Gli2 activator concentrations or when
co-transfected with Gli3 repressor (Q.L. and M.P.M., unpublished). This could
be due to a requirement for co-factors [such as Fu or Su(Fu)] that are known
to regulate Gli activity through binding to the N-terminal region deleted from
our constructs (Murone et al.,
2000), or to other modulators of Gli activator activity that
function specifically in the ventral spinal cord.
Our studies do not address the mechanisms that are responsible for
Shh/Gli-mediated progenitor segregation. In principle, regulation of either
differential cell adhesion or proliferation rates, or some combination of the
two, could be involved. In Drosophila wing imaginal discs, Hh
signaling in anterior (A) cells controls their segregation from posterior (P)
cells at the AP border (Tepass et al.,
2002). Inactivation of Hh or Ci result in mutant cells exhibiting
sorting behavior characteristic of P cells in which Hh signaling is normally
blocked by En (Dahmann and Basler,
2000
). Phenotypically similar results were obtained by
manipulating cadherin levels, providing a potential link between Hh signaling
and the control of differential cell affinity
(Dahmann and Basler, 2000
).
The striking similarities in fly and vertebrate phenotypes that result from
manipulating Hh/Ci/Gli signaling suggests that the mechanisms that control
differential cell segregation downstream of Hh signaling are conserved through
evolution.
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ACKNOWLEDGMENTS |
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Footnotes |
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