1 Developmental Genetics Program and the Department of Cell Biology, The
Skirball Institute of Biomolecular Medicine, New York University Medical
Center, 540 First Avenue, New York, NY 10016, USA
2 Harvard University, Department of Molecular and Cellular Biology, Cambridge,
Curie Building, 45 Moulton Street, Cambridge, MA 02138, USA
* Author for correspondence (e-mail: fishell{at}saturn.med.nyu.edu)
Accepted 23 July 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Telencephalon, Shh, Gli3, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Shh has been shown to play a fundamental role in the establishment of
ventral identity throughout the neural tube (reviewed by
Ericsson et al., 1995a;
Litingtung and Chiang, 2000b
;
Patten and Placzek, 2000
). As
a result, much work has been performed to elucidate the mechanisms by which
Shh generates cell diversity within the ventral neural tube, especially in the
spinal cord. A considerable body of evidence favors the idea that Shh acts as
a morphogen, forming a gradient within the ventral neural tube to which cells
respond in a concentration-dependent manner (reviewed by
Briscoe and Ericsson, 2001
;
Jessell, 2000
). Collectively,
in vitro explant studies using recombinant Shh
(Ericsson et al., 1996
;
Ericsson et al., 1997
;
Roelink et al., 1995
) and in
vivo electroporation studies with Shh
(Agarwala et al., 2001
) or a
dominant-negative form of patched (Briscoe
et al., 2001
) strongly support this hypothesis in the midbrain and
spinal cord, respectively.
Although Shh is also clearly required in forebrain regions
(Chiang et al., 1996;
Ericsson et al., 1995b
), to
date less effort has been focused on its role in patterning the telencephalon.
Similar to the spinal cord, embryos that lack Shh fail to form
ventral telencephalic structures and to express corresponding markers, while
dorsal marker expression is expanded ventrally
(Chiang et al., 1996
).
Furthermore, ectopic expression of Shh is sufficient to induce
ventral telencephalic markers in dorsal regions of the telencephalon both in
vitro (Ericsson et al., 1995b
;
Kohtz et al., 1998
;
Shimamura and Rubenstein,
1997
) and in vivo (Barth and
Wilson, 1995
; Corbin et al.,
2000
; Gaiano et al.,
1999
; Hauptmann and Gerster,
1996
). However, when and how Shh is required for ventral
telencephalic patterning and whether it acts to differentially specify the
MGE, the LGE and the CGE is not presently clear. Beginning around E9.0, Shh is
expressed within the ventral telencephalon, primarily in the MGE, preoptic
area and prospective amygdala (Nery et
al., 2001
; Sussel et al.,
1999
). Notably, the specific loss of Shh expression
within the telencephalon, which occurs in Nkx2.1 (Titf1 -
Mouse Genome Informatics) (Sussel et al.,
1999
) and BF1 (Foxg1 - Mouse Genome Informatics)
(Huh et al., 1999
) null
mutants, does not recapitulate the phenotype observed in Shh mutants.
This suggests that earlier expression of Shh, probably from non-neural tissues
such as the dorsal foregut (i.e. prechordal plate)
(Shimamura and Rubenstein,
1997
) or Hensen's node
(Gunhaga et al., 2000
), must
be responsible for the more extensive telencephalic defects seen in
Shh mutants.
The Ci/Gli family of zinc-finger transcription factors has been implicated
as required transducers of the hedgehog signaling pathway (reviewed by
Aza-Blanc and Kornberg, 1999;
Ruiz i Altaba et al., 2002
).
Gli proteins, like Ci, display both activator and repressor activities that
are regulated post-translationally
(Aza-Blanc et al., 1997
;
Ingham and McMahon, 2001
).
Different regions of the Gli proteins encode distinct functions: N-terminal
regions encode a repressor function, whereas C-terminal regions are required
for positive activity. Analysis of mice mutants for each of the three murine
Gli genes has shown that Gli3 is the only Gli gene that plays a
significant role in patterning the telencephalon. Specifically, mice that lack
Gli1 and Gli2 show minor defects in telencephalic patterning
(Park et al., 2000
), whereas
Gli3 mutant mice have strong abnormalities in the dorsal
telencephalon (Grove et al.,
1998
; Theil et al.,
1999
; Tole et al.,
2000
). Gli3 protein appears to function primarily as a repressor
(reviewed by Ingham and McMahon,
2001
) and its activity seems to be negatively regulated by Shh
(Marigo et al., 1996
;
Wang et al., 2000
). Shh
signaling, both in vitro and in the limb, has been shown to inhibit Gli3
processing into an N-terminal fragment that carries a repressor activity. In
agreement with this, analysis of mice lacking both Shh and
Gli3 revealed that, in the spinal cord, Shh is required to inhibit
Gli3 function in order to form motoneurons
(Litingtung and Chiang,
2000a
).
In the present study, we have used both gain- and loss-of-function methods
to study the importance of hedgehog signaling in general and Shh in particular
in the establishment of dorsoventral patterning in the telencephalon. We find
that virally mediated ectopic sonic hedgehog expression induces different
regional markers, depending on the dorsoventral or anteroposterior position of
the infection. Conversely, some aspects of ventral telencephalic patterning
persist in the absence of Shh. In addition, we observe that dorsoventral
patterning in the telencephalon appears to be largely restored in
Shh/Gli3 and Smo/Gli3 double homozygous mutants. Our data
reveal that Shh and Gli3 antagonize each other's function in patterning the
telencephalon. Furthermore, our findings suggest that a hedgehog-independent
mechanism, such as possibly the BMP-signaling pathway
(Liem et al., 2000), is
capable of acting in parallel to Shh in establishing dorsoventral pattern in
the telencephalon.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genotyping of mutant mice
Shh (Chiang et al.,
1996) and Gli3 (XtJ from Jackson
Laboratory) mutant mice were maintained on a C57/B16 and C3HeB/FeJ background,
respectively. Different mutant combinations were generated by intercrossing
Shh+/-;Gli3+/- or
Smo+/-;Gli3+/- heterozygous animals.
Embryos were genotyped by PCR as described previously
(Litingtung and Chiang, 2000a
;
Maynard et al., 2002
;
Zhang et al., 2001
).
Staining of tissue sections
Whole heads or embryos were fixed at 4°C for 1-4 hours in 4%
paraformaldehyde, then cryoprotected overnight in 30% sucrose in PBS, embedded
in HistoPrep (Fisher Scientific) and frozen. All tissue was sectioned serially
at 12 to 20 µm and processed for immunohistochemistry or RNA in situ
hybridization.
Infected cells were identified by the expression of the alkaline
phosphatase reporter (PLAP) as described previously
(Gaiano et al., 1999).
Analysis of gene induction was performed by RNA in situ hybridization on
adjacent 20 µm sections. Note that the thickness of the sections and the
incompatibility of histochemistry and in situ hybridization on the same
sections sometimes made it hard to correlate the infected cells with the cells
that display ectopic expression (e.g. Fig.
2J-K).
|
Immunofluorescence was performed as described previously
(Corbin et al., 2000). The
following primary antibodies were used: mouse anti-Pax6 (1:1000, gift of A.
Kawakami) and rabbit anti-Gsh2 (1:3500, gift of K. Campbell).
In situ hybridization was performed as described previously
(Schaeren-Wiemers and Gerfin-Moser,
1993; Wilkinson and Nieto,
1993
) using non-radioactive digoxigenin-labeled probes for
Nkx2.1 (Shimamura et al.,
1995
), Dlx2 (Porteus
et al., 1991
), Gsh2
(Hsieh-Li et al., 1995
),
Mash1 (Ascl1 - Mouse Genome Informatics)
(Guillemot and Joyner, 1993
),
Gli1, Gli3 (Hui et al.,
1994
) and Ptch
(Goodrich et al., 1997
).
Dlx2 mRNA expression was also determined by X-Gal staining of
embryos from a strain of mice in which the taulacZ gene has been
targeted to the Dlx2 locus
(Corbin et al., 2000) (S.
Nery, G. F. and J. G. C., unpublished). X-Gal staining was performed as
described previously (Corbin et al.,
2000
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
ActSmo- and Shh-expressing viruses were injected at various times ranging from E8.5 to E10.5 and embryos were sacrificed 4 days later for analysis (E12.5 to E14.5). Notably, we observed that the ectopic expression of ActSmo (or Shh itself) resulted in the same modifications of telencephalic patterning regardless of the age of the infection (data not shown). This suggests that the competence of the telencephalic tissue to respond to ectopic induction of the Shh pathway is not temporally regulated within this window of embryonic development.
Ventral patterning by Shh in the spinal cord is in part mediated through
the inhibition of dorsally expressed transcription factors
(Briscoe et al., 2000;
Ericsson et al., 1997
).
Similarly in the telencephalon, in vitro exposure to Shh can inhibit the
expression of dorsal markers (Kohtz et
al., 1998
). Moreover, dorsal gene expression expands ventrally in
the absence of Shh (Chiang et al.,
1996
). We found that the expression of Pax6, a dorsal
telencephalic marker, is indeed repressed in the infected cells
(Fig. 2A-C), in accordance with
previous results in spinal cord (Ericsson
et al., 1996
). A similar result was obtained for the dorsally
expressed proneural genes such as Ngn2, Math2 and NeuroD
(data not shown).
We next sought to determine the extent to which ventral markers characteristic of the MGE (Nkx2.1) and MGE/LGE (pan-ventral genes Gsh2, Dlx2) could be induced ectopically by virally mediated activation of Shh signaling. We observed that the identity of the markers induced, and therefore the cell types, was mainly dependent on the position of the infection. Along the dorsoventral axis, the induction of Nkx2.1 expression in response to ectopic Shh signaling was restricted to the LGE and lateral neocortex (Fig. 2G,H), whereas Gsh2 and Dlx2 could be induced throughout the neocortex (Fig. 2I,J and data not shown). Interestingly, we noted that the induction of pan-ventral gene expression (Gsh2, Dlx2) was weaker in the medial regions of the telencephalon (Fig. 2I,J). Sagittal sections of ActSmo infected brains revealed the rostrocaudal extent of ventral gene induction: while Nkx2.1 induction was restricted to the LGE and septum (Fig. 2D,F and data not shown), pan-ventral gene induction within the cortex was only excluded from the more posterior regions (Fig. 2D,E). These differences could be accounted for by regional variations in the ability of the telencephalon to activate downstream components of the Shh pathway. To address this issue we analyzed patched (Ptch) and Gli1 gene expression, which are believed to be generic downstream response genes in the Shh signaling pathway. We found that the expression of either of these genes was upregulated at all positions along the dorsoventral or anteroposterior axis (Fig. 2K-O). Based on these criteria, this suggests that all regions of the telencephalon are equally competent to respond to Shh signaling.
These gain-of-function experiments show that regional identity can be altered by Shh signaling through the early phases of neurogenesis. However, we found that this plasticity is somewhat limited: the ventral identities that Shh and ActSmo can induce are dependent on the dorsoventral or anteroposterior position of the mis-expression. This suggests that an intrinsic pattern, which is either Shh dependent or independent, has been established within the telencephalon by the time the Shh pathway is activated ectopically (E9.0 at the earliest, i.e. 12 hours after infection).
Pan-ventral gene expression is maintained in Shh
mutants
Previous analysis of Shh-null animals revealed that, in addition
to the lack of any obvious ventral telencephalic structures, the domain of
dorsal gene expression spreads ventrally to include the whole telencephalon
(Chiang et al., 1996;
Pabst et al., 2000
). However,
when we investigated the expression of pan-ventral genes in these mutants, we
were surprised to observe that some ventral telencephalic patterning remains
in the absence of Shh (Fig. 3).
Dlx2 expression was observed in the remaining telencephalon of E10.5
to E12.5 Shh mutants as visualized by in situ hybridization or by
using a Dlx2taulacZ allele
(Fig. 3A-D). Similarly,
Gsh2 expression (another pan-ventral gene) persisted in Shh
mutants (Fig. 3E,F) (H.
Toresson and K. Campbell, personal communication). Notably, in these mutants
the domain of expression of both Dlx2 and Gsh2 was
restricted to a region adjacent to the ventral midline. As Shh
mutants are extremely dysmorphic, it is impossible to know with certainty
which ventral telencephalic domains persist in these mutants. Nonetheless, the
fact that Nkx2.1 expression was missing in Shh-/-
embryos (data not shown) (Pabst et al.,
2000
) while Dlx2 and Gsh2 expression persisted
suggests that the most ventral domain (i.e. MGE) is absent in
Shh-/- mutants, while the lateral regions (i.e. LGE) are
displaced ventrally. In this regard, this result is likely to be analogous to
that in spinal cord, where the laterally positioned V0 and V1 interneurons,
which are characteristic of lateral cell fates, persist in Shh-/-
embryos but are displaced toward the midline
(Pierani et al., 1999
). The
observation that ventral gene expression can occur in the absence of Shh
suggests the existence of a Shh-independent pathway. This prompted us to
examine other genes that could play a role in establishing dorsoventral
patterning in the telencephalon.
|
Shh has been shown to be required to antagonize Gli3 function in ventral
regions of the spinal cord (Litingtung and
Chiang, 2000a). In the telencephalon, Gli3 is expressed
at high levels in dorsal and lateral regions (cortex and LGE) and at low
levels ventrally (in the MGE). Interestingly, extra-toes mice
(XtJ), which carry a deletion encompassing the
Gli3 gene (Buscher and Ruther,
1998
; Hui and Joyner,
1993
; Schimmang et al.,
1992
; Vortkamp et al.,
1992
), show defects opposite to those observed in Shh
mutants. In Gli3 mutants the morphology of the forebrain is perturbed
dorsally: the cortex is highly reduced in size, and the hippocampus and the
choroid plexus are absent (Grove et al.,
1998
; Theil et al.,
1999
; Tole et al.,
2000
). Furthermore, the expression of a number of pan-ventral
genes, such as, Dlx2 (Fig.
3G-H) (Tole et al.,
2000
), Gsh2 (Fig.
3I-J) and the bHLH gene Mash1 (data not shown), spreads
precociously into cortical areas. Even though ventral markers were ectopically
expressed, the cortex of Gli3 mutants retained its dorsal character,
as shown by the persistence of dorsal gene expression, including Pax6
(Fig. 3K,L)
(Tole et al., 2000
).
Interestingly, Gsh2 and Pax6 proteins appeared to be co-expressed in the
cortex of Gli3 mutants, as shown by double immunostaining
(Fig. 3L). Gsh2 and
Pax6 expression domains generally abut at the boundary between the
cortex and the LGE (i.e. the corticostriatal boundary)
(Fig. 3K) and these proteins
are normally mutually repressive (Toresson
et al., 2000
; Yun et al.,
2001
). Our results showed that Gsh2 expression no longer
respects this boundary in the telencephalon of Gli3 mutants,
suggesting that the repression of Gsh2 by Pax6 is dependent on Gli3.
Partial rescue of dorsoventral patterning in
Shh-/-;Gli3+/- mutants
In order to analyze the extent to which dorsoventral patterning can occur
in the absence of Shh and Gli3, we analyzed the
telencephalic phenotype of Shh/Gli3 compound mutants. Interestingly,
Shh-/-;Gli3+/- embryos displayed a remarkable
rescue of brain morphology when compared with Shh-/-
embryos. Although the telencephalon of Shh-/- embryos at
E12.5 was greatly reduced in size and composed of a single vesicle fused at
the midline (Fig. 4B,B')
(Chiang et al., 1996),
Shh-/-;Gli3+/- embryos had a much bigger
telencephalon in which two vesicles were clearly discernable
(Fig. 4C,C', arrows).
Moreover, these embryos had a reduced proboscis (arrowheads) and two
relatively well-formed eyes, albeit fused together and located at the midline
(Fig. 4D-S, arrows).
|
As these embryos showed a significant morphological rescue of the Shh-null phenotype, we analyzed in detail the status of dorsoventral patterning in the telencephalon. We observed that Dlx2 expression, which was shifted either ventrally or dorsally in Shh-/- and Gli3-/- mutants, respectively, was restored in Shh-/-;Gli3+/- embryos to a level and distribution which closely resembled the wild-type pattern (Fig. 4D,E). A similar rescue was observed for other pan-ventral genes such as Gsh2 and Mash1 (Fig. 4J-M). The restoration of the expression of pan-ventral genes to their normal regions was further demonstrated by the existence of a sharp boundary between the Gsh2 and Pax6 expression domains, similar to what is observed at the corticostriatal boundary of wild-type brains (Fig. 4N,O). Notably, while the telencephalic morphology of the Shh-/-;Gli3+/- embryos was affected, a thickening in the lateral wall of the telencephalon was observed ventrally to the Pax6 expression domain in a region where pan-ventral genes were strongly expressed (Fig. 4D-S; arrowheads). Thus, it seems that the LGE is properly specified in these mutants.
To further investigate to what extent dorsoventral patterning was restored in Shh-/-;Gli3+/- embryos, we looked at the expression of the homeobox gene Nkx2.1, which is missing in Shh-/- embryos. Surprisingly, we observed that low levels of Nkx2.1 expression were rescued in these embryos (Fig. 4F,G). In these animals Nkx2.1 expression was specifically located in the most ventral part of the telencephalon and was nested within the domains of expression of Dlx2 (Fig. 3H,I), Gsh2 and Mash1. Thus, although not very prominent, it appeared that a small MGE-like structure can also form in these embryos in absence of Shh (Fig. 4D-S, asterisks). The high levels of recovery observed in Shh mutants where only one copy of Gli3 gene is removed prompted us to investigate the extent of recovery in animals lacking both Shh and Gli3 gene function.
Dorsoventral patterning is largely restored in the telencephalon of
Shh/Gli3 double mutants
Telencephalic analysis of Shh/Gli3 double homozygous mutants was
extremely difficult as a result of a high rate of exencephaly. Most of the
Shh/Gli3 double homozygous mutants analyzed were exencephalic
(11/12=91%): the neural tube failed to close at various levels of the
forebrain or midbrain and a massive overgrowth of the tissue disrupted
telencephalic morphology. We found that
Shh-/-;Gli3-/- embryos had a higher proportion
of exencephaly than did Gli3-/- (2/12=16.6%) or
Gli3-/-;Shh+/- (10/32=31%) embryos. Although
exencephalic mutants could not be analyzed for dorsal patterning defects,
ventral patterning was analyzed in parallel to non-exencephalic mutants. As
the exencephalic defects were more severe at E12.5 than at E10.5, we chose to
analyze the double homozygous mutants at E10.5.
In a Shh-/-;Gli3-/- double mutant embryo that was not exencephalic, the telencephalon appeared to have a relatively normal morphology, although dorsal midline structures were missing (Fig. 5; arrows). The level and distribution of expression of pan-ventral genes such as Dlx2 and Gsh2 was similar in Shh-/-;Gli3-/- embryos and in their wild-type littermates (Fig. 5A-D). Remarkably, in contrast to Shh-/-;Gli3+/- and Shh-/- embryos, double homozygous mutants had a complete rescue of Nkx2.1 expression (Fig. 5E,F). Similar to wild-type animals, Nkx2.1 expression was observed in an area nested within the wider expression domain of Dlx2 (Fig. 5G,H, asterisk) and Gsh2 (Fig. 5C,D). This nested expression pattern suggested that both the MGE and the LGE were specified in Shh-/-;Gli3-/- mutants. However, in the absence of a more refined analysis of gene expression patterns within the ventral telencephalon, we cannot exclude the possibility that some aspects of dorsoventral patterning (such as the ventral midline) are not restored in these mutants. Nonetheless, our data suggest that a key role of Shh in patterning the ventral telencephalon is to inhibit Gli3 function, thereby indirectly allowing ventral gene expression. Complementary to the restoration of ventral patterning defects observed in Shh-/- mutants, the dorsal telencephalic defects present in Gli3-/- mutants were also rescued in Shh-/-;Gli3-/- double mutants. This argues for a requirement for Gli3 in antagonizing Shh function in the dorsal telencephalon, a role apparently not played by Gli3 in the spinal cord (Litingtung and Chiang, 2001a).
|
The persistence of dorsoventral patterning in the absence of Shh
and Gli3 gene function could be the result of compensation by other
members of the hedgehog family or the actions of a hedgehog-independent
pathway. To differentiate between these possibilities, we analyzed ventral
telencephalic gene expression in Smoothened/Gli3 double mutants, in
which all hedgehog signaling is abrogated
(Zhang et al., 2001).
Although, as in the Shh/Gli3 mutants, these animals suffer from a
high rate of exencephaly, ventral aspects of the Smo/Gli3
telencephalon can still be analyzed. In these mutants both pan-ventral markers
such as Dlx2, as well as the MGE-specific marker Nkx2.1 were
expressed in their normal distribution
(Fig. 5M,N). Furthermore, the
fact that Nkx2.1 expression was nested within the Dlx2
expression domain suggested that ventral patterning is largely rescued in
these compound mutants. Thus, our results reveal that, with the exception of
the dorsal midline, a hedgehog-independent signaling pathway is able to
establish at least basic elements of dorsoventral patterning in the
telencephalon in the absence of both Gli3 and Shh or Smo
gene function.
To determine whether the restoration of dorsoventral patterning in
Shh-/- mutants when Gli3 is removed
(Shh-/-;Gli3-/-) was due to an activation of
downstream elements of the Shh pathway independent of Shh protein, we examined
the levels of expression of Gli1 and Ptch, two targets of
Shh signaling. Gli1 and Ptch are normally expressed in the
ventral telencephalon in close proximity to the sites of Shh expression
(Platt et al., 1997).
Gli1 expression in the telencephalon is restricted to a small region
at the border of the MGE and the LGE (Fig.
4P, Fig. 5I;
brackets). As in Shh-/- mutants (data not shown) this
expression was absent in Shh-/-;Gli3+/- and
Shh-/-;Gli3-/- embryos
(Fig. 4Q,
Fig. 5J), consistent with the
suggestion that Gli1 activation is strictly dependent on Shh. In
wild-type animals Ptch is expressed in a broader region than
Gli1, encompassing the entire extent of the MGE
(Fig. 4R,
Fig. 5K; brackets). Whereas
Ptch expression is absent in Shh-/- and
Shh-/-;Gli3+/- mutants (data not shown and
Fig. 4S), low levels of
Ptch expression were detected throughout the telencephalon in
Shh-/-;Gli3-/- mutants, with higher levels
ventrally (Fig. 5L, brackets).
Higher levels of expression ventrally were also observed in double homozygous
mutants that were exencephalic (data not shown). This suggests that
Ptch expression is dependent on the removal of Gli3 repressor
activity in ventral areas and that the loss of Ptc1 expression
observed in Shh-/- mutants may be due to a lack of
inhibition of Gli3 function.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Telencephalic competence to respond to Shh signaling
In the present study, we undertook an in vivo gain-of-function approach to
address the role of Shh in telencephalic patterning. By infecting the mouse
telencephalon at different time points (E8.5 to E10.5), we showed that the
differential induction of MGE and LGE/MGE markers by Shh signaling was
determined by the intrinsic character of the infected tissues, within both the
dorsoventral and anteroposterior axes. Although it could be argued that the
failure to induce Nkx2.1 in dorsal regions of the telencephalon or
Gsh2 in dorsomedial telencephalon was the result of our viral vectors
producing insufficient levels of Shh signaling, results from our previous
studies argue against this (Kohtz et al.,
1998). Indeed, Nkx2.1 expression could not be induced in
vitro by Shh in E11.5 rat dorsal telencephalic explants (E9.5 in mouse), even
when they were exposed to extremely high levels of recombinant protein (i.e.
300 nM). Furthermore, the differential induction we observed with our viral
vectors did not appear to be the result of a differential activation of the
Shh pathway in dorsal and lateral regions, as Gli1 and Ptch
could be induced ectopically in the telencephalon, regardless of where or when
the infection occurred. Thus, our gain-of-function data suggest that by
E8.5/E9.0 the telencephalon has been patterned along both the dorsoventral and
anteroposterior axis. However, these experiments cannot distinguish whether
this apparent prepattern is the result of hedgehog independent signaling or
patterning by Shh prior to E9.0. In support of the latter suggestion, work by
the Edlund laboratory suggests that the telencephalic expression of
Nkx2.1 results from Shh signaling in the node
(Gunhaga et al., 2000
).
Similarly, we found that rat explants at headfold stage (E9.5 in rat, E8.0 in
mouse) could uniformly induce Nkx2.1 expression in response to
recombinant Shh protein (Kohtz et al.,
1998
), suggesting that the restriction observed in this paper may
not exist in slightly younger embryos (before E9.0).
Even if Shh normally acts to prepattern the telencephalon prior to E9.0,
our results show that some ventral pattern is established in the telencephalon
in absence of Shh. Specifically, we found that Dlx2 and Gsh2
expression within the ventral telencephalon is reduced but not absent in
Shh-/- mutant embryos. This result is reminiscent of the
persistence of V0 and V1 cell fates in the spinal cord of
Shh-/- mutants
(Pierani et al., 1999). Given
that in some contexts members of the hedgehog family can partially compensate
for the loss of Shh (Pathi et
al., 2001
), perhaps the persistence of ventrolateral markers
indicates the presence of other hedgehog ligands. Indeed, Indian and sonic
hedgehog are both expressed in the early foregut
(Bitgood and McMahon, 1995
;
Ingham and McMahon, 2001
).
Unfortunately, this question cannot be addressed directly as
Smo-/- mutants die before any telencephalic patterning is
molecularly evident (Zhang et al.,
2001
). However, based on the analysis of Smo/Gli3 double
homozygous mutants (see below), such compensation appears unlikely.
The regulation of ventral and lateral patterning in the telencephalon
by Shh and Gli3.
To futher investigate the cross-repressive interaction between Shh and Gli3
in the telencephalon, we analyzed several Shh and Gli3
mutant combinations. The development of the telencephalon is grossly abnormal
in Shh-/- (Chiang et
al., 1996) and to a lesser extent in Gli3-/-
mutants (Grove et al., 1998
;
Theil et al., 1999
). We found
that telencephalic morphology is largely restored in
Shh-/-;Gli3+/- mutants. Consistent with their
rescued morphology, most ventrolateral patterning is established in
Shh mutants lacking a single copy of Gli3
(Shh-/-;Gli3+/-). Furthermore, barring
the loss of dorsal midline structures, mutants lacking both Shh and
Gli3 function possess sets of markers characteristic of MGE, LGE and
cortex, showing that all these structures are at least partially specified in
the absence of Shh and Gli3. The same result was obtained in
Smo-/-;Gli3-/- mutants in which all
hedgehog signaling is abolished. Therefore, many aspects of dorsoventral
telencephalic patterning can be established in the complete absence of
hedgehog provided that Gli3 gene function is also abolished.
Despite this, the interplay between Shh and Gli3 is crucial to the normal positioning of the different domains of ventral telencephalic gene expression. For example, our data suggests that MGE and LGE markers are differentially sensitive to the dose of Gli3 protein or activity. Although the expression of pan-ventral genes characteristic of the LGE is fully restored in Shh-/- animals with one copy of Gli3 removed, the MGE marker Nkx2.1 is only rescued in the complete absence of Gli3 gene function. Consistent with this, unlike more broadly expressed ventral markers, Nkx2.1 cannot be induced by ectopic Shh signaling in the cortex, where Gli3 is highly expressed. Similarly, Nkx2.1 expression does not spread dorsally in Gli3-/- mutants, whereas pan-ventral gene expression does. One explanation for these findings may be that Gli2, which is expressed in a pattern similar to Gli3 in the telencephalon, possesses weaker repressor activity than Gli3 and acts in a complementary fashion. In this scenario, Gli2 prevents the expansion of Nkx2.1 expression in Gli3-/- mutants but is insufficient to repress pan-ventral genes, such as Dlx2 and Mash1. Indeed, the greater sensitivity of MGE markers to repression by Gli repressors may be one mechanism that underlies the differential specification of the MGE and LGE.
In the limb, the processing of Gli3 into an N-terminal repressor form is
negatively regulated by Shh (Wang et al.,
2000). As a result, Shh and the N-terminal Gli3 repressor are
thought to form opposing gradients in the limb, akin to that suggested by our
genetic analysis of the telencephalon. Our results also in many respects
parallel the phenotype found in the spinal cord of Shh/Gli3 double
mutants (Litingtung and Chiang,
2000a
). Similar to the telencephalon, ventrolateral cell fates in
the spinal cord, including motoneurons and V0-V2 interneurons, were restored
to wild-type levels in Shh/Gli3 double homozygous mutants. However,
unlike the telencephalon, the ventral-most fates fail to form in the spinal
cord of these same animals (as indicated by the loss of Nkx2.2 at the
floorplate). Moreover, only partial rescue of ventrolateral genes expression
was observed in the spinal cord of these mutants, while ventrolateral
telencephalic gene expression appeared normal in
Shh-/-;Gli3+/- mutants. Furthermore,
in the telencephalon the antagonism between Shh and Gli3 is reciprocal. By
contrast, there is no evidence that Gli3 may antagonize Shh function in the
spinal cord, as ventral gene expression does not expand dorsally in this
tissue in the absence of Gli3 gene function.
Evidence for a hedgehog independent pathway in patterning the ventral
telencephalon
In light of the Shh/Gli3 and Smo/Gli3 double mutants
phenotypes, it is worth considering how other signaling pathways might
contribute to dorsoventral telencephalic patterning. It is possible that
hedgehog-dependent and -independent pathways act in parallel and are
functionally redundant, each able to specify ventral, lateral and dorsal cell
fates in isolation. When hedgehog signaling is missing, as it is in
Shh-/-;Gli3-/- and
Smo-/-;Gli3-/- mutants, such
redundancy would account for the observed rescue of dorsoventral patterning
(Fig. 6). The use of two
pathways that complement each other is reminiscent of the mechanism of
anterior patterning in Drosophila. In flies, opposing anterior and
posterior gradients specify the segmented body pattern. Maternal nanos protein
acts as a repressor of hunchback in the posterior region, allowing abdominal
patterning to occur; and the absence of both genes results in normal embryos
(Hulskamp et al., 1989;
Irish et al., 1989
;
Struhl, 1989
). In parallel,
bicoid specifies anterior patterning and hunchback can substitute for it in
thorax and abdomen (Simpson-Brose et al.,
1994
; Wimmer et al.,
2000
).
|
Alternatively, we cannot formally exclude the possibility that the dorsoventral patterning of the telencephalon is initially established by a hedgehog-independent pathway and that the role of Shh in patterning the telencephalon is to subsequently maintain or refine the organization of the telencephalon. Perhaps Shh and Gli3, through their antagonistic interactions, act to stabilize dorsoventral telencephalic domains during mid-neurogenesis. This might occur by regulating the growth of the progenitor pools, by maintaining a pre-established pattern or by refining the regional boundaries between discrete territories, such as the MGE, LGE and cortex.
The identity of the signal(s) that lead to a normal dorsoventral patterning
in the absence of Shh and Gli3 remains unknown. One
possibility is that the dorsoventral patterning observed in Shh/Gli3
double mutants is the result of BMP signaling, which in spinal cord acts to
induce dorsal and repress ventral gene expression. Recent studies in the
spinal cord have also shown that BMPs antagonizes Shh signaling and that this
inhibition may be crucial to the establishment of dorsal identities
(Liem et al., 2000). It seems
unlikely that the late dorsal expression of BMPs contributes to
hedgehog-independent patterning as their expression requires Gli3
(Theil et al., 1999
). However,
earlier BMP7 expression in the prechordal plate or BMP4 expression in
presomitic mesoderm might be playing a role
(Dale et al., 1997
;
Dale et al., 1999
), as might
Nodal signaling (Rohr et al.,
2001
). Another alternative comes from studies in spinal cord that
have implicated the retinoid signaling pathway in the generation of
ventrolateral progenitors (Pierani et al.,
1999
). In the telencephalon, markers of retinoid synthesis are
expressed in the LGE and the developing striatum
(Li et al., 2000
;
Toresson et al., 1999
) and
retinoid signaling regulates striatal neuronal differentiation
(Toresson et al., 1999
).
However, this expression occurs too late to play a role in the initial
patterning of the telencephalon. If retinoids do act in the establishment of
dorsoventral pattern in the telencephalon, a more likely source is the lateral
cranial mesoderm, which expresses high levels of retinoids and is proximal to
the telencephalon during the headfold stage
E7.5/E8.0
(LaMantia et al., 1993
). A
third candidate is the Wnt pathway. Recent work has implicated an important
role for Wnt inhibitors in the specification of the head (dickkopf)
(Niehrs et al., 2001
) as well
as the telencephalon (masterblind)
(Heisenberg et al., 2001
;
van de Water et al., 2001
).
Furthermore, a novel secreted Frizzled-related protein expressed in the
anterior neural plate at the junction between neural and non-neural ectoderm
is involved in promoting telencephalic development within the forebrain
territory (Houart et al.,
2002
). Prior to neural tube closure, the dorsoventral axis of the
telencephalon seems to be roughly translated into an anteroposterior axis
(Shimamura et al., 1995
).
Thus, it is worth considering that, in addition to specifying the
telencephalon as a whole, graded inhibition of Wnt signaling may also act to
establish dorsoventral identity in the telencephalon.
Regardless of the pathway, it is clear that a complete appreciation of regional patterning within the telencephalon will require more than understanding the hedgehog signaling pathway. Determining the nature of these signals and how they act to complement the hedgehog pathway in patterning the telencephalon will no doubt prove interesting.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agarwala, S., Sanders, T. A. and Ragsdale, C. W.
(2001). Sonic hedgehog control of size and shape in midbrain
pattern formation. Science
291,2147
-2150.
Aza-Blanc, P. and Kornberg, T. B. (1999). Ci: a complex transducer of the hedgehog signal. Trends Genet. 15,458 -462.[CrossRef][Medline]
Aza-Blanc, P., Ramirez-Weber, F. A., Laget, M. P., Schwartz, C. and Kornberg, T. B. (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89,1043 -1053.[Medline]
Barth, K. A. and Wilson, S. W. (1995).
Expression of zebrafish nk2.2 is influenced by sonic hedgehog/vertebrate
hedgehog-1 and demarcates a zone of neuronal differentiation in the embryonic
forebrain. Development
121,1755
-1768.
Bitgood, M. J. and McMahon, A. P. (1995). Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol. 172,126 -138.[CrossRef][Medline]
Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol. Cell 7,1279 -1291.[CrossRef][Medline]
Briscoe, J. and Ericsson, J. (2001). Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11,43 -49.[CrossRef][Medline]
Briscoe, J., Pierani, A., Jessell, T. M. and Ericsson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101,435 -445.[Medline]
Buscher, D. and Ruther, U. (1998). Expression profile of Gli family members and Shh in normal and mutant mouse limb development. Dev. Dyn. 211, 88-96.[CrossRef][Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Corbin, J. G., Gaiano, N., Machold, R. P., Langston, A. and
Fishell, G. (2000). The Gsh2 homeodomain gene controls
multiple aspects of telencephalic development.
Development 127,5007
-5020.
Dahmane, N. and Ruiz-i-Altaba, A. (1999). Sonic
hedgehog regulates the growth and patterning of the cerebellum.
Development 126,3089
-3100.
Dale, J. K., Vesque, C., Lints, T. J., Sampath, T. K., Furley, A., Dodd, J. and Placzek, M. (1997). Cooperation of BMP7 and SHH in the induction of forebrain ventral midline cells by prechordal mesoderm. Cell 90,257 -269.[Medline]
Dale, K., Sattar, N., Heemskerk, J., Clarke, J. D., Placzek, M.
and Dodd, J. (1999). Differential patterning of ventral
midline cells by axial mesoderm is regulated by BMP7 and chordin.
Development 126,397
-408.
Ericsson, J., Muhr, J., Jessell, T. M. and Edlund, T. (1995a). Sonic hedgehog: a common signal for ventral patterning along the rostrocaudal axis of the neural tube. Int. J. Dev. Biol. 39,809 -816.[Medline]
Ericsson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T. (1995b). Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81,747 -756.[Medline]
Ericsson, J., Morton, S., Kawakami, A., Roelink, H. and Jessell, T. M. (1996). Two critical periods of Sonic Hedgehog signaling required for the specification of motor neuron identity. Cell 87,661 -673.[Medline]
Ericsson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, A., van Heyningen, V., Jessell, T. M. and Briscoe, J. (1997). Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling. Cell 90,169 -180.[Medline]
Fishell, G. (1997). Regionalization in the mammalian telencephalon. Curr. Opin. Neurobiol. 7, 62-69.[CrossRef][Medline]
Gaiano, N., Kohtz, J. D., Turnbull, D. H. and Fishell, G. (1999). A method for rapid gain-of-function studies in the mouse embryonic nervous system. Nat. Neurosci. 2, 812-819.[CrossRef][Medline]
Goodrich, L. V., Milenkovic, L., Higgins, K. M. and Scott, M.
P. (1997). Altered neural cell fates and medulloblastoma in
mouse patched mutants. Science
277,1109
-1113.
Grove, E. A., Tole, S., Limon, J., Yip, L. and Ragsdale, C.
W. (1998). The hem of the embryonic cerebral cortex is
defined by the expression of multiple Wnt genes and is compromised in
Gli3-deficient mice. Development
125,2315
-2325.
Guillemot, F. and Joyner, A. L. (1993). Dynamic expression of the murine Achaete-Scute homologue Mash-1 in the developing nervous system. Mech. Dev. 42,171 -185.[CrossRef][Medline]
Gunhaga, L., Jessell, T. M. and Edlund, T.
(2000). Sonic hedgehog signaling at gastrula stages specifies
ventral telencephalic cells in the chick embryo.
Development 127,3283
-3293.
Hauptmann, G. and Gerster, T. (1996). Complex
expression of the zp-50 pou gene in the embryonic zebrafish brain is altered
by overexpression of sonic hedgehog. Development
122,1769
-1780.
Heisenberg, C. P., Houart, C., Take-Uchi, M., Rauch, G. J.,
Young, N., Coutinho, P., Masai, I., Caneparo, L., Concha, M. L., Geisler, R.
et al. (2001). A mutation in the Gsk3-binding domain of
zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon
and eyes to diencephalon. Genes Dev.
15,1427
-1434.
Houart, C., Caneparo, L., Heisenberg, C., Barth, K., Take-Uchi, M. and Wilson, S. (2002). Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron 35,255 -265.[Medline]
Hsieh-Li, H. M., Witte, D. P., Szucsik, J. C., Weinstein, M., Li, H. and Potter, S. S. (1995). Gsh-2, a murine homeobox gene expressed in the developing brain. Mech. Dev. 50,177 -186.[CrossRef][Medline]
Huh, S., Hatini, V., Marcus, R. C., Li, S. C. and Lai, E. (1999). Dorsalventral patterning defects in the eye of BF-1-deficient mice associated with a restricted loss of shh expression. Dev. Biol. 211,53 -63.[CrossRef][Medline]
Hui, C. C. and Joyner, A. L. (1993). A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene. Nat. Genet. 3,241 -246.[Medline]
Hui, C. C., Slusarski, D., Platt, K. A., Holmgren, R. and Joyner, A. L. (1994). Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev. Biol. 162,402 -413.[CrossRef][Medline]
Hulskamp, M., Schroder, C., Pfeifle, C., Jackle, H. and Tautz, D. (1989). Posterior segmentation of the Drosophila embryo in the absence of a maternal posterior organizer gene. Nature 338,629 -632.[CrossRef][Medline]
Hynes, M., Ye, W., Wang, K., Stone, D., Murone, M., Sauvage, F. and Rosenthal, A. (2000). The seven-transmembrane receptor smoothened cell-autonomously induces multiple ventral cell types. Nat. Neurosci. 3,41 -46.[CrossRef][Medline]
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signaling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
Irish, V., Lehmann, R. and Akam, M. (1989). The Drosophila posteriorgroup gene nanos functions by repressing hunchback activity. Nature 338,646 -648.[CrossRef][Medline]
Jensen, A. M. and Wallace, V. A. (1997).
Expression of Sonic hedgehog and its putative role as a precursor cell mitogen
in the developing mouse retina. Development
124,363
-371.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Kalderon, D. (2000). Transducing the hedgehog signal. Cell 103,371 -374.[Medline]
Kohtz, J. D., Baker, D. P., Corte, G. and Fishell, G.
(1998). Regionalization within the mammalian telencephalon is
mediated by changes in responsiveness to Sonic Hedgehog.
Development 125,5079
-5089.
LaMantia, A. S., Colbert, M. C. and Linney, E. (1993). Retinoic acid induction and regional differentiation prefigure olfactory pathway formation in the mammalian forebrain. Neuron 10,1035 -1048.[Medline]
Li, H., Wagner, E., McCaffery, P., Smith, D., Andreadis, A. and Drager, U. C. (2000). A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse. Mech. Dev. 95,283 -289.[CrossRef][Medline]
Liem, K. F., Jr, Jessell, T. M. and Briscoe, J.
(2000). Regulation of the neural patterning activity of sonic
hedgehog by secreted BMP inhibitors expressed by notochord and somites.
Development 127,4855
-4866.
Litingtung, Y. and Chiang, C. (2000a). Specification of ventral neuron types is mediated by an antagonistic interaction between Shh and Gli3. Nat. Neurosci. 3, 979-985.[CrossRef][Medline]
Litingtung, Y. and Chiang, C. (2000b). Control of Shh activity and signaling in the neural tube. Dev. Dyn. 219,143 -154.[CrossRef][Medline]
Marigo, V., Johnson, R. L., Vortkamp, A. and Tabin, C. J. (1996). Sonic hedgehog differentially regulates expression of GLI and GLI3 during limb development. Dev. Biol. 180,273 -283.[CrossRef][Medline]
Maynard, T. M., Jain, M. D., Balmer, C. W. and LaMantia, A. S. (2002). High-resolution mapping of the Gli3 mutation extra-toes reveals a 51.5-kb deletion. Mamm. Genome 13, 58-61.[CrossRef][Medline]
Murone, M., Rosenthal, A. and de Sauvage, F. J. (1999). Hedgehog signal transduction: from flies to vertebrates. Exp. Cell Res. 253,25 -33.[CrossRef][Medline]
Nery, S., Wichterle, H. and Fishell, G. (2001).
Sonic hedgehog contributes to oligodendrocyte specification in the mammalian
forebrain. Development
128,527
-540.
Niehrs, C., Kazanskaya, O., Wu, W. and Glinka, A. (2001). Dickkopf1 and the Spemann-Mangold head organizer. Int. J. Dev. Biol. 45,237 -240.[Medline]
Pabst, O., Herbrand, H., Takuma, N. and Arnold, H. H. (2000). NKX2 gene expression in neuroectoderm but not in mesendodermally derived structures depends on sonic hedgehog in mouse embryos. Dev. Genes Evol. 210,47 -50.[Medline]
Park, H. L., Bai, C., Platt, K. A., Matise, M. P., Beeghly, A.,
Hui, C. C., Nakashima, M. and Joyner, A. L. (2000). Mouse
Gli1 mutants are viable but have defects in SHH signaling in combination with
a Gli2 mutation. Development
127,1593
-1605.
Pathi, S., Pagan-Westphal, S., Baker, D. P., Garber, E. A., Rayhorn, P., Bumcrot, D., Tabin, C. J., Blake Pepinsky, R. and Williams, K. P. (2001). Comparative biological responses to human Sonic, Indian, and Desert hedgehog. Mech. Dev. 106,107 -117.[CrossRef][Medline]
Patten, I. and Placzek, M. (2000). The role of Sonic hedgehog in neural tube patterning. Cell Mol. Life Sci. 57,1695 -1708.[Medline]
Pierani, A., Brenner-Morton, S., Chiang, C. and Jessell, T. M. (1999). A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord. Cell 97,903 -915.[Medline]
Platt, K. A., Michaud, J. and Joyner, A. L. (1997). Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice. Mech. Dev. 62,121 -135.[CrossRef][Medline]
Porteus, M. H., Bulfone, A., Ciaranello, R. D. and Rubenstein, J. L. (1991). Isolation and characterization of a novel cDNA clone encoding a homeodomain that is developmentally regulated in the ventral forebrain. Neuron 7,221 -229.[Medline]
Roelink, H., Porter, J. A., Chiang, C., Tanabe, Y., Chang, D. T., Beachy, P. A. and Jessell, T. M. (1995). Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis. Cell 81,445 -455.[Medline]
Rohr, K. B., Barth, K. A., Varga, Z. M. and Wilson, S. W. (2001). The nodal pathway acts upstream of hedgehog signaling to specify ventral telencephalic identity. Neuron 29,341 -351.[Medline]
Rowitch, D. H., St-Jacques, B., Lee, S. M., Flax, J. D., Snyder,
E. Y. and McMahon, A. P. (1999). Sonic hedgehog regulates
proliferation and inhibits differentiation of CNS precursor cells.
J. Neurosci. 19,8954
-8965.
Rubenstein, J. L. and Beachy, P. A. (1998). Patterning of the embryonic forebrain. Curr. Opin, Neurobiol. 8,18 -26.[CrossRef][Medline]
Ruiz i Altaba, A., Palma, V. and Dahmane, N. (2002). Hedgehog-Gli signaling and the growth of the brain. Nat. Rev. Neurosci. 3,24 -33.[CrossRef][Medline]
Schaeren-Wiemers, N. and Gerfin-Moser, A. (1993). A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes. Histochemistry 100,431 -440.[Medline]
Schimmang, T., Lemaistre, M., Vortkamp, A. and Ruther, U.
(1992). Expression of the zinc finger gene Gli3 is affected in
the morphogenetic mouse mutant extra-toes (Xt).
Development 116,799
-804.
Shimamura, K., Hartigan, D. J., Martinez, S., Puelles, L. and
Rubenstein, J. L. (1995). Longitudinal organization of the
anterior neural plate and neural tube. Development
121,3923
-3933.
Shimamura, K. and Rubenstein, J. L. (1997).
Inductive interactions direct early regionalization of the mouse forebrain.
Development 124,2709
-2718.
Simpson-Brose, M., Treisman, J. and Desplan, C. (1994). Synergy between the hunchback and bicoid morphogens is required for anterior patterning in Drosophila. Cell 78,855 -865.[Medline]
Struhl, G. (1989). Differing strategies for organizing anterior and posterior body pattern in Drosophila embryos. Nature 338,741 -744.[CrossRef][Medline]
Sussel, L., Marin, O., Kimura, S. and Rubenstein, J. L.
(1999). Loss of Nkx2.1 homeobox gene function results in a
ventral to dorsal molecular respecification within the basal telencephalon:
evidence for a transformation of the pallidum into the striatum.
Development 126,3359
-3370.
Theil, T., Alvarez-Bolado, G., Walter, A. and Ruther, U.
(1999). Gli3 is required for Emx gene expression during dorsal
telencephalon development. Development
126,3561
-3571.
Tole, S., Ragsdale, C. W. and Grove, E. A. (2000). Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J). Dev. Biol. 217,254 -265.[CrossRef][Medline]
Toresson, H., Mata de Urquiza, A., Fagerstrom, C., Perlmann, T.
and Campbell, K. (1999). Retinoids are produced by glia in
the lateral ganglionic eminence and regulate striatal neuron differentiation.
Development 126,1317
-1326.
Toresson, H., Potter, S. S. and Campbell, K.
(2000). Genetic control of dorsal-ventral identity in the
telencephalon: opposing roles for Pax6 and Gsh2.
Development 127,4361
-4371.
van de Water, S., van de Wetering, M., Joore, J., Esseling, J.,
Bink, R., Clevers, H. and Zivkovic, D. (2001). Ectopic Wnt
signal determines the eyeless phenotype of zebrafish masterblind mutant.
Development 128,3877
-3888.
Vortkamp, A., Franz, T., Gessler, M. and Grzeschik, K. H. (1992). Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt). Mamm. Genome 3,461 -463.[Medline]
Wang, B., Fallon, J. F. and Beachy, P. A. (2000). Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb. Cell 100,423 -434.[Medline]
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron 22,103 -114.[Medline]
Wichterle, H., Turnbull, D. H., Nery, S., Fishell, G. and
Alvarez-Buylla, A. (2001). In utero fate mapping reveals
distinct migratory pathways and fates of neurons born in the mammalian basal
forebrain. Development
128,3759
-3771.
Wilkinson, D. G. and Nieto, M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 225,361 -373.[Medline]
Wilson, S. W. and Rubenstein, J. L. (2000). Induction and dorsoventral patterning of the telencephalon. Neuron 28,641 -651.[Medline]
Wimmer, E. A., Carleton, A., Harjes, P., Turner, T. and Desplan,
C. (2000). Bicoid-independent formation of thoracic segments
in Drosophila. Science
287,2476
-2479.
Xie, J., Murone, M., Luoh, S. M., Ryan, A., Gu, Q., Zhang, C., Bonifas, J. M., Lam, C. W., Hynes, M., Goddard, A. et al. (1998). Activating Smoothened mutations in sporadic basal-cell carcinoma. Nature 391,90 -92.[CrossRef][Medline]
Yun, K., Potter, S. and Rubenstein, J. L.
(2001). Gsh2 and Pax6 play complementary roles in dorsoventral
patterning of the mammalian telencephalon. Development
128,193
-205.
Zhang, X. M., Ramalho-Santos, M. and McMahon, A. P. (2001). Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node. Cell 106,781 -792.[CrossRef][Medline]