Download Combinatorial Gli gene function in floor plate and

2203
Development 125, 2203-2212 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV6336
Combinatorial Gli gene function in floor plate and neuronal inductions by
Sonic hedgehog
A. Ruiz i Altaba
The Skirball Institute, Developmental Genetics Program and Department of Cell Biology, NYU Medical Center, 540 First Avenue,
New York, NY 10016, USA
*Author for correspondence: (e-mail: [email protected])
Accepted 2 April; published on WWW 19 May 1998
SUMMARY
Within the developing vertebrate nervous system, it is not
known how progenitor cells interpret the positional
information provided by inducing signals or how the
domains in which distinct groups of neural cells
differentiate are defined. Gli proteins may be involved in
these processes. In the frog neural plate, we have previously
shown that the zinc finger transcription factor Gli1 is
expressed in midline cells and mediates the effects of Shh
inducing floor plate differentiation. In contrast, Gli2 and
Gli3 are expressed throughout the neural plate except for
the midline. Here, it is shown that Gli3 and Shh repress
each other whereas Gli2, like Gli1, is a target of Shh
signaling. However, only Gli1 can induce the differentiation
of floor plate cells. In addition, Gli2 and Gli3 repress the
ectopic induction of floor plate cells by Gli1 in co-injection
assays and inhibit endogenous floor plate differentiation.
The definition of the floor plate domain, therefore, appears
to be defined by the antagonizing activities of Gli2 and Gli3
on Gli1 function. Because both Gli1 and Gli2 are induced
by Shh, these results establish a regulatory feedback loop
triggered by Shh that restricts floor plate cells to the
midline. We have also previously shown that the Gli genes
induce neuronal differentiation and here it is shown that
there is specificity to the types of neurons the Gli proteins
induce. Only Gli1 induces Nkx2.1/TTF-1+ ventral forebrain
neurons. Moreover, Gli2 and Gli3 inhibit their
differentiation. In contrast, the differentiation of spinal
motor neurons can be induced by the two ventrally
expressed Gli genes, Gli1 and Gli2, suggesting that Gli2
directly mediates induction of motor neurons by Shh. In
addition, Gli3 inhibits motor neuron differentiation by
Gli2. Thus, combinatorial Gli function may pattern the
neural tube, integrating positional information and cell
type differentiation.
INTRODUCTION
To analyze the molecular mechanisms involved in the
interpretation of positional information, we have focused on
the function of the Gli gene family (Kinzler et al., 1987;
Ruppert et al., 1988, 1990; Walterhouse et al., 1993; Hui et al.,
1994; Marigo et al., 1996; Lee et al., 1997; Marine et al., 1997;
Platt et al., 1997; Hughes et al., 1997) which includes cubitus
interruptus (ci; Orenic et al., 1990; Eaton and Kornberg, 1990)
in Drosophila. In frog embryos, Gli1 is expressed in midline
cells at the time that they are induced to become floor plate and
in cells lateral to the midline that appear to differentiate into
primary motor neurons (Lee et al., 1997). Gli2 is expressed
throughout the neural plate with the exception of the midline
and Gli3, also absent from the midline, shows a graded
expression with highest levels laterally. We have shown that
Gli1, but not Gli3, is a target of Shh and that it can mediate
floor plate induction by Shh (Lee et al., 1997). Consistent with
these results, Gli1 also induces ectopic ventral development in
mice (Hynes et al., 1997) and mimics Shh signaling in the
chick limb (Marigo et al., 1996). Gli proteins show a conserved
five zinc finger domain with highest identity in the last three
fingers, which bind DNA (Pavletich and Pabo, 1993). Indeed,
A critical question in understanding the formation of the
vertebrate nervous system is how the mechanisms of positional
information interface with the mechanisms of neurogenesis and
gliogenesis to yield a patterned program of neural cell
differentiation. Within the early frog neural plate, this problem
can be defined as how the floor plate attains its identity and how
its domain is restricted to the midline, whereas distinct types of
neurons differentiate at defined positions within the neural plate.
Mediolateral (M-L), future dorsoventral (D-V), pattern
develops partly as the result of positional information
provided by the graded and antagonizing actions of Sonic
hedgehog (Shh), from the notochord and floor plate (Riddle
et al., 1993; Echelard et al., 1993; Krauss et al., 1993; Roelink
et al., 1994, 1995; Ruiz i Altaba et al., 1995; Ekker et al.,
1995; Martí et al., 1995; Ericson et al., 1995, 1996; Hynes et
al., 1995; Chiang et al., 1996), and Bone Morphogenetic
Proteins (BMPs), derived initially from the inducing
epidermal ectoderm (Moury and Jacobson, 1989; Liem et al.,
1995; Dickinson et al., 1995).
Key words: Gli, Sonic hedgehog, Floor plate, Induction, Pattern
formation, Gene function, Neuron, Zinc finger
2204 A. Ruiz i Altaba
Gli proteins recognize the same target sites in vitro (Kinzler
and Vogelstein, 1990; Vortkamp et al., 1995; Sasaki et al.,
1997; Marine et al., 1997). Finally, all three Gli proteins have
a strong neurogenic effect and Gli function is involved in
primary neurogenesis (Brewster et al., 1998).
Here, the effects of Gli proteins on neural patterning are
examined and it is shown that the Gli genes respond to Shh and
induce distinct programs of neural cell differentiation. The
results suggest that the definition of the domains of floor plate
and ventral forebrain neuronal differentiation involve a
regulatory feedback loop in which Gli2, which is induced by
Shh at a distance from midline cells, acts to restrict the activity
of Gli1 to a ventral domain. In addition, Gli2 appears to mediate
the induction of motor neurons by Shh. The restriction of motor
neuron differentiation by Gli2 to the ventral region may be due
to an inhibitory effect of Gli3, the expression of which overlaps
with that of Gli2 in dorsal regions. Together, these results
suggest that the Gli proteins interact in partially overlapping and
partially antagonizing ways to establish spatial pattern and cell
identity in the developing vertebrate nervous system.
RNA probes were made as follows: a Shh cDNA clone (Ruiz i Altaba
et al., 1995) was digested with NotI and transcribed with T3. A Pax3
cDNA clone (Kim et al., 1997) was digested with EcoRI and
transcribed with SP6. An xHB9 cDNA (Saha et al., 1997) was digested
with NotI and transcribed with T7. RNA probes for frog and mouse
Gli1, Gli2 and Gli3 and mouse Shh were as described (Lee et al.,
1997).
Labeling frog embryos with anti-HNF-3β (Ruiz i Altaba et al.,
1995; Lee et al., 1997) or anti-Nkx2.1/TTF-1 polyclonal antibodies
(Lazzaro et al., 1991) by whole-mount immunocytochemistry with
peroxidase-DAB/H2O2 was as described (Lee et al., 1997).
Histological sections (10-15 µm) of Paraplast-embedded embryos
were cut in a microtome and mounted in Permount.
RESULTS
The Gli genes respond to Shh signaling
Gli1 is expressed in midline neural plate cells (Fig. 1A) and then
in immediately adjacent cells (Fig. 1B), which appear to be early
differentiating primary motor neurons (Lee et al., 1997). In
contrast, Gli2 and Gli3 are expressed widely in the neural plate
but not at the midline (Fig. 1C,E), with Gli3 showing a graded
MATERIALS AND METHODS
Embryos and microinjection
Xenopus laevis embryos were obtained and reared by standard
techniques and staged according to Nieuwkoop and Faber (1967).
Microinjection of plasmid DNA (100-200 pg/10 nl/embryo) or
synthetic RNA was performed by standard techniques (e.g. Ruiz i
Altaba, 1993) into one cell at the 1- to 2-cell stage. RNA injections were
at 2 ng/10 nl/embryo unless otherwise indicated. Co-injections were
performed at 1 ng for each RNA (2 ng total), and single injections
serving as controls for the co-injections were performed at 1 ng/embryo
also thus maintaining the same amount of tested RNA (e.g. Gli1 or
Gli2). Albino embryos were routinely used for injection. For mouse
embryos E0.5 was counted as the morning when the plug was found.
Plasmid clones
The vectors used for injection into frog embryos were pcDNA1-Amp
(Invitrogen) or pCS2 and pCS2-myc tag (Turner and Weintraub, 1994;
Rupp et al., 1994). All vectors used contained CMV regulatory
elements, which provide ubiquitous expression of human (h) or frog
(f) cDNAs. Plasmids expressing hGli1, hGli3 and fGli1 were as
described previously (Lee et al., 1997). Plasmids driving the
expression of fShh were as described in Ruiz i Altaba et al. (1995).
The hGli1-hGli3 chimeric construct was made by replacing the Nterminal region (bp 1-1248) of hGli3 with a 702 bp Pwo PCR Nterminal fragment of hGli1, in pcDNA1-Amp. The swap point was
immediately upstream of the zinc finger DNA-binding domain in both
hGli1(at bp 702, aa 208) and hGli3 (at bp 1248, aa 398). The internal
zinc finger deletion of hGli3 was obtained by linearization with BglII
followed by controlled digestion with Bal31 and religation. The inframe deletion (504-643) removes the zinc finger DNA-binding
domain with the exception of the first 22 aa of finger 1. fGli2 and
fGli3 cDNAs derived from our initial Gli screen (Lee et al., 1997) and
were as described (Brewster et al., 1998).
Synthetic RNAs for injection were made by transcription of pCS2
constructs with SP6 polymerase after digestion with NotI in the
presence of cap analog. Reactions were done at 30°C to enhance
production of full-length 4-5 kb transcripts.
In situ hybridization and immunocytochemistry
Whole-mount in situ hybridization was carried out as described by
Harland (1991) using maleic acid. Antisense digoxigenin-labeled
Fig. 1. Neural Gli gene expression, induction of Gli2 and repression
of Gli3 by Shh. (A,B) Expression of Gli1 in midline (md) cells of a
late gastrula/early neurula embryo (A; stage ~12) and in immediately
adjacent cells in older neurulae (B; stage ~14). (C) Expression of
Gli2 in a neural plate stage embryo (stage 13; n=45). Note its
absence from the midline. (D) Ectopic expression of Gli2 (arrows) in
a stage ~13 embryo injected with Shh plasmids (88% n=34). Ectopic
expression of Gli2 was also induced by hGli1 (27% n=11) or VP16fGli1 (38% n=21; not shown). (E) Expression of Gli3 at the early
neurula stage (stage ~13, n=23) in the neural folds (nf). Inset shows
cross section of a stage 14 embryo showing a graded medial to lateral
expression. (F) Expression of Gli3 in an embryo (stage ~13) injected
with Shh plasmids (32% n=40) with unilateral repression of Gli3
(arrows). Repression of Gli3 was also observed after injection of
hGli1 (20% n=20) or fGli1 (30% n=10; not shown).
(A) Lateral/posterior view; (B,C,E,F) dorsal views with anterior end
up; (D) lateral view with dorsal side to the left and anterior end up.
Dorsal side is up for the section in the inset of E. Dashed line depicts
the axis of bilateral symmetry. md, midline cells.
Pattern formation by combinatorial Gli gene function 2205
distribution along the M-L axis (Fig. 1E inset). Because Gli1 is
a target of Shh in the neural plate (Lee et al., 1997) and Gli3 is
repressed by Shh in the chick limb (Marigo et al., 1996), we
tested for the effects of Shh on the expression of Gli2 and Gli3.
Frog embryos injected with plasmids driving the expression of
Shh displayed the ectopic, albeit mosaic, expression of Gli2 in
the ectoderm (Fig. 1D). In contrast, Shh repressed Gli3
transcription (Fig. 1F). Similar results were obtained by injecting
Gli1 (not shown), consistent with the role of Gli1 as a mediator
of Shh signaling (Lee et al., 1997).
Gli3 mutant embryos display ectopic Shh
expression in the dorsal neural tube
The ability of Shh to repress Gli3 in the neural plate together
with the ectopic expression of Shh in anterior limb regions of
Gli3 mutant mice (Masuya et al., 1995), raised the possibility
that this mutual antagonistic interaction between Gli3 and Shh
is also operative in the neural tube. In the spinal cord of E12.5
mouse embryos, Shh is expressed in the floor plate (Fig. 2A),
Gli1 is expressed in the ventral ventricular zone immediately
dorsal to the floor plate (Fig. 2C; Lee et al., 1997; Sasaki et al.,
1997) and Gli2 and Gli3 are expressed in the dorsal ventricular
zone, with Gli2 also showing low levels of expression in the
ventral ventricular zone (Fig. 2D and not shown; Lee et al.,
1997; Sasaki et al., 1997). Extra-toes (XtJ) mutant mice have
an intragenic deletion of Gli3 and show overt defects in brain
development (Hui and Joyner, 1993; Franz, 1994). E12.5 extratoes mutant embryos were found to display moderate levels of
ectopic Shh expression in the dorsal ventricular zone of the
neural tube, the region that normally expresses Gli2 and Gli3
(Fig. 2C and not shown: Hui et al., 1994; Lee et al., 1997;
Sasaki et al., 1997). Little change was observed in XtJ mice
for Gli2 expression whereas Gli1 was ectopically expressed at
low levels (not shown). Ectopic gene expression in XtJ mice,
however, is a variable phenotype and was detected in under
30% of tested homozygote −/− embryos. Such variability has
also been reported for marker expression in the limb buds of
Xt mice (Masuya et al., 1995). The near normal morphology
of the spinal cord in some Gli3 mutant mice suggests that other
factors, in addition to Gli3, regulate Shh. Nevertheless, Gli3
appears to be a general repressor of Shh.
Table 1. Quantitation of injection results
(A) Ectopic induction of HNF-3β+ cells by Gli1 but not Gli2 or Gli3
pDNA
Marker
Injection
Ectopic
Normal
n
HNF-3β
"
"
"
"
"
"
fGli1
hGli1
fGli2
fGli3
hGli3
hGli1→hGli3
control
98
72
5
0
6
49
2
2
28
95
100
94
51
98
100
190
40
30
170
72
42
(B) Antagonistic action of Gli2 and Gli3 on ectopic HNF-3β induction by
Gli1
Marker
Injection
Ectopic Normal
n
pDNA
HNF-3β
"
"
"
"
"
"
"
"
hGli1
" + hGli3
" + hGli3zf∆
" + fGli2
45
6
60
17
55
94
40
83
42
150
148
53
fGli1
" + hGli3
" + hGli3zf∆
" + fGli2
60
28
40
24
40
72
60
76
60
28
50
66
(C) Differential effects of Gli proteins on endogenous HNF-3β expression
RNA
Fig. 2. Expression of Shh, Gli1 and Gli2 in wild-type mouse embryos
and ectopic Shh expression in Gli3 mutant embryos. (A) Expression
of Shh RNA in the floor plate (fp) in the thoracic spinal cord of wildtype (+/+) E12.5 mice. (B) Ectopic expression of Shh in the dorsal
ventricular zone of E12.5 Gli3 mutant (−/−) mice. The ectopic
expression of Shh in the ventricular zone mimics the normal
expression of Gli2 (D) and Gli3 (Lee et al., 1997; Sasaki et al.,
1997). (C) Expression of Gli1 in the spinal cord of E12.5 wild-type
mice. Note the focal expression in the ventral ventricular zone (vvz).
(D) Expression of Gli2 in a wild-type E12.5 spinal cord. High levels
are present in the dorsal ventricular zone (dvz) and lower and more
restricted expression is detected in the ventral ventricular zone (vvz).
All panels show cross sections with dorsal side up.
Marker
Injection
Ectopic
Repressed
Normal
n
HNF-3β
"
"
"
"
"
fGli1
fGli2
fGli3
hGli3
" D2/4
hGli3-zf∆
100
0
0
0
0
0
0
14
20
50
81
0
0
86
80
50
19
100
18
14
14
12
32
45
(D) Reciprocal rescue of the effects of Glis on HNF-3β expression
RNA
Marker
Injection
HNF-3β
"
"
"
"
"
fGli1
" + fGli3
" + hGli3
" + hGli3zf∆
" + fGli2
control
Ectopic
Repressed
Normal
n
100
16
19
93
23
0
0
0
14
0
4
0
0
84
67
7
73
100
48
42
47
46
52
61
(A-D) Summary of injection results as indicated. All numbers refer to
percentages except the total number of embryos (n). D2/4 in C signifies
injection into both dorsal blastomeres at the 4-cell stage. See text for details.
2206 A. Ruiz i Altaba
Gli1, but not Gli2 or Gli3, can induce floor plate cell
differentiation
To test the effects of injected Gli proteins (Table 1A), the
expression of HNF-3β has been used as a marker of floor plate
cell differentiation in early tadpoles (Fig. 3A; Ruiz i Altaba et
al., 1995; Lee et al., 1997). Expression of Gli1 proteins from
injected plasmids or synthetic RNA resulted in ectopic
expression of HNF-3β within the neural tube (Fig. 3B,C; Table
1B,D). Neither injection of Gli2 nor Gli3 resulted in ectopic
HNF-3β expression (Table 1B,D; Lee et al., 1997). These
results show that Gli proteins have non-redundant functions as
only Gli1 can induce floor plate differentiation.
Gli proteins appear to recognize the same target
genes in vivo
Because both human Gli1 and Gli3 bind to the same DNA
sequences in vitro (Kinzler and Vogelstein, 1990; Vortkamp et
al., 1995; Sasaki et al., 1997), a chimeric Gli3 protein was
constructed having its N-terminal region replaced by that of
Gli1, retaining the zinc finger DNA-binding and C-terminal
domains of Gli3 (Table 1A). Injection of this hGli1→hGli3
chimera resulted in ectopic expression of HNF-3β, a Gli1 target
gene, albeit at lower frequency than hGli1 (Table 1B). The Nterminal part of hGli1 can therefore confer floor-plate-inducing
activity to hGli3 and suggests that Gli proteins can recognize
the same DNA targets in vivo.
Gli2 and Gli3 antagonize ectopic floor plate
induction by Gli1
If all Gli proteins bind the same targets, the inability of Gli2 and
Gli3 to activate HNF-3β indicates that they could act as
repressors in vivo, consistent with the repressor activity of Gli3
in vitro (Marine et al., 1997; Sasaki et al., 1997). To investigate
whether Gli2 or Gli3 could repress Gli1 function, plasmids
driving the expression of Gli1 and Gli3 or Gli1 and Gli2 were
co-injected into developing frog embryos. Gli2 and Gli3 were
able to repress the ectopic HNF-3β-inducing activity of frog or
human Gli1 (Table 1C), a nuclear-targeted Gli1 and a VP16-Gli1
derivative (not shown; Lee et al., 1997). Injection of a Gli3
mutant construct with an in-frame deletion of the zinc finger
DNA-binding domain was ineffective (Table 1C). Together,
these results show that Gli2 and Gli3 lack the ability to induce
floor plate development and that they antagonize the effects of
Gli1, possibly by competition for regulatory sites in target genes.
Gli2 and Gli3 repress endogenous floor plate
development
The effects of ectopic Gli3 and Gli2 expression in the ventral
neural tube were examined in light of their ability to repress
the effects of co-injected Gli1 in vivo and the fact that their
expression domains partially overlap (Lee et al., 1997).
Injection of Gli2 or Gli3 RNAs into the dorsal quadrant of early
frog embryos resulted in embryos displaying axial defects as
compared to uninjected sibling controls (Fig. 3D). Within the
ventral neural tube, injection of Gli2 or Gli3, but not a Gli3
zinc finger deletion mutant, resulted in the loss of HNF-3β
from ventral neural tube cells (Fig. 3F,N; Table 1D), normally
overlying the notochord (Fig. 3E,M). The expression of Shh
mRNA was also analyzed in Gli3-injected embryos as a second
ventral marker. Within the neural tube of early tadpoles, Shh is
normally expressed in the ventrolateral diencephalon, the
intrathalamic region and the floor plate (Fig. 3G,I; Ruiz i
Altaba et al., 1995; Ekker et al., 1995). As with HNF-3β,
embryos injected with Gli1 showed ectopic Shh expression
(Lee et al., 1997) and those injected with Gli3 showed loss of
its ventral expression in the floor plate and diencephalon (Fig.
3H,J), suggesting that Gli3 inhibits the differentiation of
several, if not all, ventral cell types. The stronger effects in
anterior regions reflects the prevalent localization of the
injected proteins to this region after injection into the animal
hemisphere and to the late development of posterior structures.
All embryos scored displayed normal notochord differentiation
as assessed morphologically or by gene/antigen expression.
Cellular differentiation that is independent of Shh was not
impeded in injected embryos. HNF-3β+/Shh+ neurons in the
intrathalamic region/midbrain were observed in Gli3-injected
embryos (Fig. 3H and not shown), consistent with their presence
in u.v.-treated notochordless frog embryos (not shown).
Moreover, Pax3 (Kim et al., 1997) was expressed at similar
levels in the dorsal neural tube in control and injected embryos
(Fig. 3K,L; n=8). Indeed, embryos ectopically expressing Gli3
in the dorsal neural tube after animal pole plasmid injections
show normal morphological development (Lee et al., 1997).
As with plasmid injections (Table 1C), coinjection of Gli1
plus Gli2 or Gli1 plus Gli3 RNAs, but not Gli1 plus a Gli3 zinc
finger deletion mutant, resulted in a dramatic decrease in the
incidence of ectopic HNF-3β expression (Table 1E). In
addition, coinjected embryos also showed a marked decrease
in the incidence of loss of endogenous HNF-3β. Thus, whereas
Gli2 and Gli3 can antagonize the effects of Gli1 on ectopic
floor plate induction, Gli1 can rescue their repression of
endogenous floor plate differentiation.
The differentiation of Nkx2.1/TTF-1+ ventral forebrain
neurons is induced by Gli1 and repressed by Gli2
and Gli3
Floor plate cells do not differentiate in the forebrain. Shh and
Gli1, however, are expressed in anterior ventral regions of the
neural tube raising the possibility that, in these regions, Gli1
may induce the differentiation of distinct classes of Shhresponsive ventral neurons. To test this possibility, the
expression of the Nkx2.1/TTF-1 homeoprotein (Lazzaro et al.,
1991) was analyzed as a marker of ventral telencephalic and
diencephalic secondary neurons (Fig. 4A,B). Nkx2.1/TTF-1 is
induced by Shh in explant culture (Ericson et al., 1995) and is
ectopically expressed in the dorsal forebrain in Gli1-injected
embryos (Fig. 4C; Lee et al., 1997). Injection of either Gli3 or
Gli2 failed to induce ectopic Nkx2.1/TTF-1+ neurons.
Moreover, both were able to repress their normal
differentiation (Fig. 4D,E and not shown). Gli3-injected
embryos also showed the loss of the ventrolateral stripe of Shh
expression in the diencephalon (Fig. 3H). Together, these
results suggest that, in the forebrain, as in more posterior
regions, Gli1 induces ventral cell differentiation whereas Gli2
and Gli3 not only lack this ability, but antagonize Gli1 function
and the differentiation of Gli1-inducible ventral cell types.
Motor neuron induction by Gli1 and Gli2
The repressive effect of Gli2 on Nkx2.1/TTF-1+ neurons
contrasts with its involvement in endogenous neurogenesis
(Brewster et al., 1998). In frog embryos, there are few specific
markers described for neurons located at distinct D-V positions
Pattern formation by combinatorial Gli gene function 2207
of the neural tube. The homeobox gene HB9, however, provides
a clean marker of secondary spinal motor neurons in the tailbud
and tadpole neural tube (Fig. 5A-C; Saha et al., 1997). Because
Gli2 is expressed in the ventral region where motor neurons
originate (Lee et al., 1997), the idea that Gli2 induces motor
neuron development was tested. Gli2-injected tadpoles (stage
~32) displayed the ectopic expression of HB9 whereas control
embryos showed only normal expression (Fig. 5F,G). Ectopic
motor neurons were observed in the dorsal hindbrain and spinal
cord but not in the forebrain or midbrain. Gli1 also induced
ectopic HB9 expression (Fig. 5D,E). This result, however, is
expected as injected Gli1 induces Gli2 expression. Embryos
injected with frog (n=14) or human (n=23) Gli3 did not display
ectopic HB9 expression (not shown). Ectopic HB9 expression
was sometimes observed in epidermal ectoderm adjacent to the
neural tube (not shown), indicating that Gli1/2 function can
induce neuronal differentiation in non-neural ectoderm. This is
consistent with previous findings demonstrating the neurogenic
activity of Gli proteins (Brewster et al., 1998). The ability of Gli2
to induce motor neuron, but not floor plate development,
together with its induction by Shh signaling, suggests that Gli2
mediates the Shh induction of motor neurons and possibly other
ventral neuronal types.
Gli3 represses motor neuron induction by Gli1 and
Gli2
Gli2 is expressed throughout the neural plate, with the
exception of the midline, and in dorsal as well as ventral
regions in the ventricular zone of the neural tube (Lee et al.,
1997; Figs 1C, 2D). Motor neurons, however, do not develop
dorsally. If the Gli readout of a cell is important for
determining cell type (Ruiz i Altaba, 1997), Gli3 acting as a
repressor dorsally is predicted to account for the ventral
restriction of the motor-neuron-inducing activity of Gli2. To
test this possibility, synthetic Gli RNAs were co-injected and
the embryos assayed for HB9 expression at the tadpole stage.
Injection of frog Gli1 resulted in the induction of motor
neurons (80% ectopic, n=5, Fig. 5) as did injection of frog Gli2
(79% ectopic, n=14, Fig. 5). In contrast, coinjection of frog
Gli1 and frog or human Gli3 resulted in a marked inhibition of
ectopic HB9 expression (50% ectopic, n=4 and 28% ectopic,
n=18, respectively). Similarly, frog and human Gli3 were able
to inhibit motor neuron induction by Gli2 (12% ectopic, n=17
and 4% ectopic, n=24, respectively). As with floor plate
induction by Gli1, human Gli3 was more potent than frog Gli3
as a repressor of motor neuron differentiation. Ectopic
expression of Gli3 in the ventral neural tube inhibited
endogenous HB9 expression (n=5; not shown), although it is
not clear whether this is a direct effect or mediated through the
inhibition of endogenous floor plate differentiation (see Fig.
3N). Together, these results indicate that Gli3 inhibits floor
plate and ventral forebrain neuron induction by Gli1 as well as
motor neuron induction by Gli1 and Gli2, consistent with the
role of Gli3 as a general repressor of Shh induction.
DISCUSSION
Gli proteins induce different programs of floor plate
and neuronal development
An unresolved question in vertebrate neural development is
how progenitor cells choose a fate induced by Shh. Gli proteins
are good candidates to participate in this process. In the frog
neural plate, Gli1 is expressed in midline and immediately
adjacent cells, responds to Shh signaling and induces floor
plate cells and ventral neuronal types (Lee et al., 1997). An
involvement of Gli1 in the differentiation of floor plate cells is
suggested by three lines of evidence. First, all Gli proteins
appear to recognize the same target sites. Second, co-injected
Gli2 or Gli3 suppress ectopic floor plate induction by Gli1.
Third, ectopic ventral expression of Gli2 or Gli3 suppresses
endogenous floor plate and ventral forebrain neuronal
differentiation. It remains possible, however, that injected Gli2
and Gli3 have additional effects that are distinct from the
repression of Gli1 function.
Gli1 may have a more general role in gliogenesis than just
induction of floor plate cells as in the mouse spinal cord Gli1
is expressed in the ventral ventricular zone (Lee et al., 1997;
Sasaki et al., 1997; Fig. 2) where oligodendrocyte precursors
are found (Noll and Miller, 1993; Yu et al., 1994). A role of
Gli1 in gliogenesis would be consistent with its initial isolation
from a glioma line (Kinzler et al., 1987).
Gli2, like Gli1, is induced by Shh but it is not expressed in
midline cells. Gli2 may respond to low doses of Shh, with high
levels at the midline repressing it. Alternatively, other midline
factors may repress Gli2 expression. Notwithstanding how
Gli2 is regulated, it is unable to induce floor plate development
but it may mediate instead the induction of neuronal types
located at a distance from the midline. This is supported by its
neurogenic function (Brewster et al., 1998), the ectopic
differentiation of HB9+ motor neurons in Gli2-injected
embryos and the ability of co-injected Gli3 to inhibit ectopic
motor neuron induction by Gli2. Gli1 also induces motor
neurons although it may be indirect through its intermediate
induction of floor plate cells and thus of Gli2. In the dorsal
neural tube, the expression and function of Gli2 is likely to be
independent of Shh suggesting that Gli2 responds to multiple
cues.
In contrast to Gli1 and Gli2, Gli3 is repressed by Shh and
may be involved in the differentiation of Shh-independent
dorsal neuronal types. A role for Gli3 in the dorsal neural tube
is supported by its neurogenic activity (Brewster et al., 1998)
and its requirement for normal development in mice and
humans (Vortkamp et al., 1991; Hui and Joyner, 1993; Franz
et al., 1994; Kang et al., 1997).
Combined and antagonizing Gli gene function
Gli proteins may balance each other’s functions in partially
overlapping domains to create pattern. In this case, the ‘Gli
readout’ of a cell is predicted to be critical for determining its
fate (Ruiz i Altaba, 1997).
Shh/Gli1 repress Gli3. At early gastrula stages prior to the
onset of Shh expression in notochord precursors, low levels of
Gli1 and Gli3 are detected in the dorsal animal cap, the
prospective neural plate before midline differentiation (Fig.
6A; Lee et al., 1997). As the notochord begins to express Shh
and the midline begins to express floor plate markers, Gli3
expression is absent from the midline, consistent with its
repression by Shh from the forming notochord (Fig. 6B,C). A
first step in ventralizing the medial neural plate by Shh may
therefore be the repression of Gli3. Studies on limb
development further suggest that Shh/Gli1 and Gli3 have
2208 A. Ruiz i Altaba
mutually repressive relationships.
Ectopic Shh expression in the
anterior chick limb bud induces
Gli1 and represses Gli3 (Marigo et
al., 1996), whereas in Gli3 mutant
mouse embryos, this region displays
ectopic Shh expression (Masuya et
al., 1995; Büscher et al., 1997).
Similarly, Gli3 represses Shh/Gli1
in the dorsal neural tube. This
implicates Gli3 and Shh/Gli1 in a
mutually repressive interaction that
appears to be critical for pattern
formation in different tissues. Gli3
must be absent for ventral cell type
induction and patterning and Gli3 is
involved in repressing Shh in dorsal
regions, thus allowing dorsal
development.
Gli2 and Gli3 have redundant
functions in repressing floor plate
induction by Gli1. However, Gli2
and Gli3 are differently regulated
and have different functions in
neuronal patterning as only Gli2 can
induce spinal motor neurons. A
partial redundancy between Gli2
and Gli3 has also been found in
mouse skeletal patterning (Mo et al.,
1997). Because the mutation
introduced in the mouse Gli2 gene
leaves intact the N-terminal region
and the first two zinc fingers, it
remains possible that such a
mutation is not a null but rather a
hypomorph if the N-terminal part of
Gli proteins were to encode a
repressive function like their
Drosophila counterpart (Aza-Blanc
et al., 1997).
A regulatory feedback loop
triggered by Shh may
determine the identity and
extent of the floor plate
Gli2 represses the floor-plateinducing function of Gli1 and
endogenous
floor
plate
differentiation. Gli2 could therefore
normally restrict the ability of Gli1 to
induce floor plate development in
cells immediately adjacent to the
midline. This interaction could
provide a molecular basis for the
spatial restrictions to the propagation
of floor plate induction previously
observed (Ruiz i Altaba et al., 1995)
that occurs before neural cells lose
their competence to respond to floorplate-inducing signals (Placzek et al.,
1993). Shh signaling from the
Fig. 3. Gli1 induces and Gli2 and Gli3 repress floor plate development. (A) Normal expression of
HNF-3β in the floor plate (fp), the midbrain (m) and zona limitans intrathalamica (zli) in a tadpole
stage (stage ~34) embryo. The telencephalon (t) lacks expression. nt, notochord. (B,C) Ectopic
expression of HNF-3β in tadpoles driven by injected hGli1 plasmids (B) resulting in a mosaic pattern,
or synthetic fGli1 RNA (C), resulting in a more generalized expression. Arrows point to sites of
ectopic expression. (D) Overall morphology of tadpoles (stage~34) injected with hGli3 RNA (bottom
row) as compared with a normal sibling (top). Similar phenotypes were observed in fGli3 and fGli2
RNA-injected embryos (not shown). Injected embryos show deficiencies in the axis, head and face.
(E,F) Normal expression of HNF-3β in the floor plate (fp) (E) and its loss in the anterior area of a
sibling embryo injected with Gli3 (F, arrow). nc, notochord; nt, neural tube. (G,I) Expression of Shh
in tadpole stage (stage ~34) embryos (n=32). (G) Shh is detected in the floor plate (fp), underlying
notochord (nc), midbrain and zona limitans intrathalamica (zli/mb) and ventrolateral diencephalon
(vdi). Shh is also expressed in the frontonasal process (fnp), pharyngeal endoderm/prechordal plate
area (pp), branchial arches (ba) and hypochord. The cement gland is unlabeled and its position ventral
to the frontonasal process is indicated. (I) high magnification of the trunk with expression of Shh in
floor plate cells. (H,J) Expression of Shh in Gli3-injected tadpoles (stage ~34). (H) Expression of Shh
in the floor plate and ventrolateral diencephalon is missing (arrows; 53%; n=17) whereas other sites
appear normal taking into account the distortion of these embryos. (J) High magnification of the
hindbrain region in which anterior floor plate cells are missing (arrow) but posterior Shh+ floor plate
cells are present. Cells in the zli/m also express Shh (see above). The level of expression of Shh in the
notochord in normal and injected embryos is similar. (K,L) Expression of Pax3 in the dorsal neural
tube of both control (K; n=25) and Gli3 RNA-injected (L; 100%; n=14) tadpoles (stage~34). Pax3 is
not expressed in the dorsal telencephalon. Both control and injected embryos also showed expression
in the trigeminal ganglion and somites (not shown). cg, cement gland; dnt, dorsal neural tube; fb,
forebrain. (M,N) Cross sections through the hindbrain of a normal embryo (M) and a sibling Gli3
RNA-injected embryo (N). HNF-3β expression within the neural tube (nt) in the nuclei of floor plate
(fp) cells overlying the notochord (nc) is absent in Gli3-injected embryos which also display a thin
ventral neural tube indicative of loss of ventral cell differentiation.
Pattern formation by combinatorial Gli gene function 2209
notochord may thus initiate a regulatory cascade by inducing
Gli1 and Gli2 expression in different yet overlapping domains.
The identity of midline cells as floor plate may be determined
by Gli1 in the absence of Gli2. In contrast, the mediolateral
(dorsoventral) extent of the floor plate may be determined by
Gli2 acting to antagonize the floor-plate-inducing function of
Gli1. This interaction would occur in Gli1+/Gli2+ cells adjacent
to the Gli1+ /Gli2− floor plate (Fig. 6B,C).
The idea that Shh initiates a feedback regulatory loop that is
critical for the patterning of induced cell types has a parallel
with another signaling system. Recent experiments show that
a class of inhibitory Smad proteins (Dad, Smad6, Smad7)
prevents TGF-β superfamily signaling by competing with
transducing Smads (e.g. Mad, Smad1, Smad2) for receptor
binding (Tsuneizumi et al., 1997, Imamura et al., 1997, Nakao
et al., 1997; Hayashi et al., 1997) or association with the
general partner Smad4 (Hatta et al., 1998). Interestingly, the
expression of Dad in Drosophila (Tsuneizumi et al., 1997) and
Smad7 in mammalian cells (Nakao et al., 1997) is induced by
Dpp and TGF-β signaling, respectively. Thus, inhibition of
signaling by induced repressors that are members of the same
family as the signal transducing molecules may be a general
mechanism that provides temporal and/or spatial patterning
information.
Combinatorial function of Gli genes in neuronal
patterning
Different Gli proteins induce different types of neurons.
Whereas Gli1 can induce a variety of ventral neuronal types
including ventral forebrain neurons, hindbrain serotonergic
neurons (Lee et al., 1997) and spinal motor neurons, Gli2 can
only induce a subset of these ventral neuronal classes.
However, in the posterior CNS, Gli1 may induce many
ventral neuronal types through the intermediate induction of
floor plate cells, which themselves will express Shh, thus
inducing Gli2 in adjacent cells. Because Gli1 is restricted to
midline and adjacent ventral cells, Gli2 is predicted to be
normally involved in inducing secondary motor neuron
differentiation in response to Shh. How then can Gli2 induce
motor neurons only in the ventral region? Two strategies
appear to play a role in restricting the motor-neuron-inducing
Fig. 5. Motor neuron induction by Gli1 and Gli2.
Expression of the homeobox gene HB9 in control
(A-C) or injected (D-G) embryos at the tadpole stage
(stage ~30-34). (A-C) Normal expression of HB9 in
bilateral pools of motor neurons in the spinal cord
(n=34) viewed dorsally (A), laterally (B) and in cross
section through the cervical region (C). (D,F)
Unilateral ectopic expression of HB9 in Gli1-injected
embryos (91%, n=22) viewed in whole mount dorsally
(D) or in cross section through the cervical spinal cord
(F). (E,G) Ectopic expression of HB9 in Gli2-injected
embryos (76%, n= 17) viewed laterally (E) and in
cross section through the cervical region (G). Note that
embryos were injected into one cell at the 2-cell stage
and thus only one half of the neural tube is affected
(denoted by a dashed line in F,G). In A,B,D,E, anterior
is to the left. In C,F,G, dorsal is on top. de, dorsal
endoderm; fp, floor plate; hb, hindbrain; mn, motor
neurons; n, notochord; p, pituitary; sc, spinal cord.
Arrows depict sites of ectopic expression.
Fig. 4. Differential effects of Gli proteins on Nkx2.1/TTF-1+ ventral
forebrain neurons. (A) Localization of Nkx2.1/TTF-1+ neurons in the
ventral diencephalon (vdi) and ventral telencephalon (vt) of a tadpole
(stage ~34; n=22). The antibody also recognizes a perinotochord (nc)
antigen. (B,C) Normal (B) and ectopic (C) expression of
Nkx2.1/TTF-1 in the forebrain of control and Gli1-injected embryos,
respectively. The panels show forward views from the midbrain into
the forebrain. Note the normal expression of Nkx2.1/TTF-1 in the
ventral diencephalon (B) and its unilateral expansion to the dorsal
forebrain (arrows in C) after injection of fGli1 RNA (78%, n=13).
The dashed line depicts the axis of bilateral symmetry. (D,E) Loss of
Nkx2.1/TTF-1 expression in the forebrain of Gli3-injected embryos.
Arrows point to the anteroventral forebrain (fb). Note the normal
differentiation of the notochord (nc) and the presence of a neural
tube (nt) devoid of Nkx2.1/TTF-1 reactivity. Loss of Nkx2.1/TTF-1
expression was observed in fGli2 RNA-injected embryos (31% n=13;
not shown), fGli3 RNA-injected embryos (43% n=14) and hGli3
RNA-injected embryos (not shown, 78% n=13). The posterior
notochord is out of focus in these micrographs.
function of Gli2 to the ventral neural tube. Ventrally, even
though Gli2 is activated by Shh, midline factors repress its
expression in prospective floor plate cells. Dorsally, Gli2 and
Gli3 expression overlaps and, at least in equivalent amounts,
Gli3 represses the motor-neuron-inducing function of Gli2.
Thus, Gli2 is transcriptionally repressed in midline cells and
2210 A. Ruiz i Altaba
A
Gli1
Gli3
Animal cap
B
Neural plate
Neural tube
Gli3
Gli2
Gli1
M
L
Gli1
Gli2
Gli3
V
D
C
Notochord
Shh
?
Gli1
Gli3
Neural plate
Shh
Fig. 6. Schematic diagram of Gli gene function and its regulation by
Shh. (A) Diagram showing that, in the blastula and early gastrula
animal cap, Gli3, but not Gli1, is expressed (Lee et al., 1997). Here,
Gli3 is proposed to repress Gli1 and Shh. (B) Diagram representing
the expression domains of the Gli genes in the neural plate (top) and
in the ventricular zone of the neural tube (bottom). Only one half of
the neural plate or neural tube is shown and the neural tube is
represented as opened dorsally and flattened out to have a direct
comparison with the neural plate. The x axis denotes mediolateral
(M-L) position in the neural plate and dorsoventral (D-V) position in
the neural tube. The y axis represents expression levels of the Gli
genes. (C) During gastrulation and as the notochord underlies the
midline of the neural plate, Shh secreted initially from the notochord
induces expression of Gli1 in medial cells and that of Gli2 in more
lateral cells. At the same time, it represses Gli3 transcription in a
graded manner. Gli1 in medial cells induces floor plate (FP)
development. Adjacent to the floor plate, Gli1 expression overlaps
that of Gli2 where very low levels of Gli3 may also be present. These
cells do not become floor plate but instead appear to be
differentiating neurons (Lee et al., 1997). Gli2, and Gli3, may inhibit
Gli1 from inducing floor plate differentiation in cells adjacent to the
midline. Here Gli2 may induce the differentiation of ventral neurons
(VN). The neurogenic abilities of Gli2 and Gli3 are also depicted by
arrows and the inhibition of the motor-neuron-inducing ability of
Gli2 by Gli3 is shown with a T bar. In dorsal regions combined
Gli2/Gli3 function may induce dorsal neurons (DN). Factors
inducing the dorsal expression of Gli3 and Gli2 are not known (?).
Arrows represent positive actions and T bars repressive actions.
Gli2
FP
VN
FP
VN
DN
Neural plate
Notochord
functionally repressed in dorsal cells, leaving only ventral
non-midline cells free of Gli2 inhibitors. Gli3 cannot induce
ventral neuronal types and is predicted to induce intermediate
and/or dorsal neurons.
It is likely that Gli proteins may interact and synergize
with other factors to create the fine pattern of neuronal types
that arise in any one region of the neural tube. It remains
possible that the graded distribution of Gli3 overlapping that
of Gli2 may result in distinct Gli combinations that induce
different neuronal fates. Testing this idea, however, awaits
the availability of unambiguous markers of different cell
types within the frog neural tube as well as determining
whether Gli proteins, like Ci (Aza-Blanc et al., 1997), can
yield varying forms with different activities. Posttranslational modification of Gli2 by Shh is hinted by the
finding that Shh signaling is required in dividing motor
neuron precursors up until late in the S phase of the last cell
cycle before post-mitotic neurons are generated (Ericson et
al., 1996). Thus, earlier transcriptional activation of Gli2 by
Shh may be a requirement for motor neuron differentiation
but it may not be sufficient. It is possible that the existence
of motor-neuron-inducing Gli2 forms is dependent on
sustained Shh signaling.
In the forebrain, Gli2 and Gli3 also inhibit Gli1 function and
Gli2 may normally induce the differentiation of a subset of
ventral neurons. Gli2, however, is unable to induce ectopic
differentiation of Nkx2.1/TTF-1+ neurons suggesting that, in
this case, Gli1 acts directly. Independently of which classes of
forebrain neurons Gli2 may induce, its repressive action on
Nkx2.1/TTF-1+ cells raises the possibility that in the forebrain,
like in more posterior CNS regions, a regulatory feedback loop
triggered by Shh patterns the ventral region. More dorsally, the
same kinds of interactions between Gli2 and Gli3 proposed for
the hindbrain and spinal cord may also be involved in neuronal
patterning.
Gli gene function in the neural plate, and in the ventricular
zone of the neural tube, may thus resemble the function of gap
genes (Rivera-Pomar and Jäckle, 1996) during Drosophila
anteroposterior patterning in that they may act to convert
gradients into stripes.
I thank Jeff Lee, Rachel Brewster, Nadia Dahmane, Gord Fishell,
Ruth Lehmann, Ed Ziff and Will Talbot for discussion and/or
comments on the manuscript. I am grateful to N. Dahmane for the
mouse in situs, E. Storm for E12.5 Extra-toes homozygote mutant
embryo bodies, A. Joyner for XtJ breeding mice, R. Di Lauro for the
anti-TTF-1/Nkx2.1 antibody, M. Saha for the xHB9 cDNA and J. Lee
for initial technical assistance. This work was supported by a Skirball
Institute start-up grant, a Basil O’Connor award from the March of
Dimes, a Pew Fellowship in the Biomedical Sciences and a grant from
the NIH (RO1-NS 37352.01).
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