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First published online 14 May 2008
doi: 10.1242/dev.015990
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Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 511, New York, NY 10021, USA.
* Author for correspondence (e-mail: joynera{at}mskcc.org)
Accepted 16 April 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Anterior-posterior patterning, Dorsal-ventral patterning, Isthmic organizer, Mid/hindbrain, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Zervas et al., 2005). Sonic
hedgehog (Shh), expressed in the notochord and the ventral midline (floor
plate) of the neural tube, controls specification of ventral cell types in the
tegmentum and formation of normal dorsal structures, as well as cell
proliferation and survival (Agarwala et
al., 2001; Bayly et al.,
2007; Blaess et al.,
2006; Corrales et al.,
2006; Fedtsova and Turner,
2001; Ishibashi and McMahon,
2002). It remains to be addressed how these two signaling centers
are integrated to control growth and patterning in three dimensions.
The zinc-finger transcription factor Gli3 influences multiple signaling
pathways in the mes/r1. Gli3 is an essential downstream component of the Shh
signaling pathway, and an increase in Gli3 repressor (Gli3R) levels is the
primary cause of the dorsal mes/r1 defects in Shh signaling mutants
(Blaess et al., 2006).
Furthermore, Gli3 plays a role in regulating the Fgf8 expression
domain at the mes/r1 boundary (Aoto et al.,
2002). Therefore, Gli3 could play a key role in coordinating
three-dimensional patterning of the mes/r1 by affecting these two signaling
pathways.
Only one mutation in mouse that is thought to represent a Gli3-
null allele has been characterized. The lethal Gli3 extratoe
(Xt) mutation causes multiple defects, including a
background-dependent occurrence of exencephaly
(Hui and Joyner, 1993;
Maynard et al., 2002). In
non-excencephalic embryos, however, tectum- and cerebellum-like structures
develop, but are morphologically highly abnormal
(Aoto et al., 2002;
Blaess et al., 2006). The
analysis of Gli3Xt/Xt mutants has so far provided little
insight into Gli3 function in the mes/r1 and it remains unclear whether the
defects result from changes in the expression of key DV patterning genes,
abnormal growth and/or changes in Fgf8 signaling in the isthmic organizer
(Aoto et al., 2002).
Gli3 transcription and protein activity are regulated at many levels by Shh
signaling. In the absence of Shh activity, Gli3 is processed into an
N-terminal repressor form that suppresses Shh target genes
(Hu et al., 2006;
Wang et al., 2000). Shh
signaling attenuates the level of Gli3R, and full-length Gli3 can act as a
weak activator (Gli3A) when the concentration of Shh is high
(Bai et al., 2004;
Dai et al., 1999;
Wang et al., 2007). Gli3,
however, is also transcriptionally downregulated in cells receiving high
levels of Shh. Consequently, Gli3 expression, which is initially
found throughout the neural plate, is restricted to the intermediate and
dorsal neural tube after E8.5 (Bai et al.,
2002; Blaess et al.,
2006; Hui et al.,
1994; Marigo et al.,
1996).
Activation of Shh target genes is primarily mediated by Gli1 and Gli2
(Fuccillo et al., 2006;
Jacob and Briscoe, 2003). Gli2
requires Shh signaling to act as an activator (Gli2A), whereas Gli1 is a
transcriptional target of Gli2/3A and is itself a constitutive activator
(Fuccillo et al., 2006;
Jacob and Briscoe, 2003). We
recently showed that Gli2A-mediated Shh signaling is the key regulator of the
initial specification of ventral neurons in the embryonic mes/r1 before E11,
and of granule cell precursor proliferation in the postnatal cerebellum
(Blaess et al., 2006;
Corrales et al., 2006;
Corrales et al., 2004). It is
unclear, however, whether Gli3A is also required for the development of these
two regions.
Analysis of the severely abnormal Gli3Xt/Xt null mutants can only provide insight into the first crucial early developmental function of Gli3. To study the underlying mechanisms of Gli3 function we combined the analysis of null and time-specific conditional mutants by generating a Gli3 conditional allele and inactivating Gli3 in the mes/r1 at either E9.0 or E11.5. We demonstrate that Gli3 is required to pattern dorsal mes/r1 into distinct structures before E9.0, continues to regulate the growth of the tectum and the cerebellum as well as cerebellar foliation between E9.0 and E11.0, and plays a role beyond E11.0 in regulating growth of the isthmus, SC and IC. We further show that the role of Gli3 in isthmic and cerebellar development, but not tectal patterning, is largely mediated through the repression of Fgf8 expression in r1.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Three targeted W4 (Auerbach et al.,
2000) ES cell clones were identified
(Matise et al., 2000) by
Southern blot analysis using SphI and a 1 kb external probe 3'
of the SphI site, or using KpnI and an internal probe
containing neo. ES cell chimeras were generated through injection of
C57BL/6 blastocysts (Skirball Transgenic Facility)
(Papaioannou and Johnson,
2000). Chimeras were bred to C57BL/6 mice and heterozygous
Gli3flox-neo/+ offspring to SV129 ACTB-Flpe mice
(Rodriguez et al., 2000) to
produce Gli3flox/+ heterozygotes lacking neo.
Gli3rec/+ mice carrying a loxP-mediated deletion were
intercrossed or crossed with Gli3Xt/+ mice
(Hui and Joyner, 1993). Both
Gli3rec/rec and Gli3rec/Xt E18.5
embryos displayed the same brain and limb phenotypes described in
Gli3Xt/Xt embryos (see Fig. S1D-F in the supplementary
material) (Hui and Joyner,
1993). Other phenotypes observed in Gli3Xt/Xt
embryos, such as lethality at birth and exencephaly in some mutants, were also
observed (data not shown). The Gli3flox allele was
genotyped using the following primers: S1,
5'-CTGGATGAACCAAGCTTTCCATC-3' and AS3,
5'-CTGCTCAGTGCTCTGGGCTCC-3'. For detecting the recombined allele,
primers were S1 (as above) and AS2,
5'-CAGTAGTAGCCTGGTTACAG-3'.
Other alleles were genotyped as described: Cre, Smoflox,
Smorec (Blaess et al.,
2006), Fgf8 null (Chi
et al., 2003) and Gli3Xt null
(Maynard et al., 2002).
Gli3Xt/+ mice were maintained on a C57/BL6 background; all
other mouse lines were maintained on an outbred Swiss Webster background. Noon
of the day a vaginal plug was observed was designated as E0.5; the day of
birth was designated as P0.
Histology, immunohistochemistry and RNA in situ hybridization
Embryos/brains were fixed in 4% paraformaldehyde (PFA) at 4°C,
processed for paraffin- or cryosectioning and sectioned at 7-12 µm.
Antibody staining, BrdU labeling and RNA in situ hybridization were performed
using standard methods. Primary antibodies: rabbit anti-TH (Chemicon, 1:500);
rabbit anti-Ki67 (NovoCastra, 1:500); mouse anti-BrdU (BD-Bioscience, 1:100);
rabbit anti-phosphohistone H3 (PH3) (1:500, Cell Signaling Technology); rabbit
anti-caspase 3 (Cell Signaling Technology, 1:200); mouse anti-calbindin
(Sigma, 1:1000); rabbit anti-calbindin (Swant, 1:5000); rabbit anti-Pax2
(Zymed, 1:500); rabbit anti-neurogranin (Chemicon, 1:500); mouse anti-Nkx6.1
[(Pedersen et al., 2006)
Developmental Studies Hybridoma Bank, 1:100]. Apoptosis was quantified by
immunostaining for cleaved caspase 3. Proliferation and/or cell cycle exit was
quantified by PH3 immunostaining or BrdU pulse labeling (1 hour or 24 hours)
followed by BrdU, BrdU/Ki67 immunostaining. Coronal sections of wild-type and
conditional mutants (
3 embryos for each genotype and stage) were used. In
situ hybridization and/or immunostaining for region-specific markers (En1 for
mes/r1 at E9.5; Otx2 for mes; Pax7 for dorsal mes/r1; Pax6 for diencephalon)
was performed on adjacent sections. Cells were counted in the ventral and
dorsal mes and r1 (3 sections) at E9.5 and E10.5 and either normalized for
ventricle length (cleaved caspase 3, PH3) or total cell number (BrdU). At
E12.5, cells in the dorsal mes (3 sections) and in r1 and ventral mes (1-3
sections) were counted and normalized for ventricle length (PH3) or cell
number (BrdU+, Ki67+/BrdU+). Cell counts were
performed using ImageJ.
Detailed protocols are available at http://www.mskcc.org/mskcc/html/77387.cfm.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sgaier et al., 2007),
En1-Cre was used to remove Gli3 specifically in the mes/r1
by E9.0, about 36 hours after the onset of Gli3 expression
(Hui et al., 1994;
Kimmel et al., 2000;
Li et al., 2002). To assess
whether Gli3 is required for mes/r1 development after midgestation,
Nestin-Cre was used to remove Gli3 from the entire neural
tube around E11.5 (Blaess et al.,
2006; Graus-Porta et al.,
2001; Tronche et al.,
1999). Indeed, inactivation of Gli3 by E9
(Gli3flox/Xt;En1-Cre mice, referred to as
En1-Gli3 cko) or E11.5 (Gli3flox/Xt;Nestin-Cre
mice, referred to as Nes-Gli3 cko) resulted in rescue of the early
postnatal lethality of Gli3-null mutants and the conditional knockout
mice survived for several months. The Gli3 conditional mutants were first compared with Gli3-null mutants at E18.5, as the latter die at birth. Gli3-null mutants (Gli3Xt/Xt, Gli3Xt/rec or Gli3rec/rec) showed a phenotype of a variable nature in the midbrain and cerebellum at E18.5 (see Fig. S2A-J in the supplementary material), but the variability was independent of the allele (rec or Xt). Therefore, all three genotypes were used to represent the Gli3-null (Gli3-/-) mutant phenotype. At the gross morphological level, the phenotype of E18.5 Gli3-/- mutants included: (1) a poorly foliated cerebellum that was not clearly separated from the isthmus; (2) an expanded isthmus-like region with ectopic cell clusters; (3) an overgrown tectum; (4) loss of the distinct morphology that normally defines the isthmus, IC and SC (8/11) (Fig. 1A,B,E,F; see Fig. S2A-J in the supplementary material). By contrast, the tegmentum of Gli3-/- mutants was morphologically unaffected and the size of the ventral nuclei comprising tyrosine hydroxylase (TH)-positive dopaminergic neurons or Isl1-positive motoneurons appeared similar to that of wild-type (WT) mice (Fig. 1I,J and data not shown).
Similar to Gli3-/- mutants, E18.5 En1-Gli3 cko mutants had a normal tegmentum, and a misshapen and enlarged tectum and isthmus (Fig. 1C,G,K; see Fig. S2K,L in the supplementary material). In contrast to Gli3-/- mutants, however, the isthmus, IC and SC each appeared as morphologically distinct structures in the majority of En1-Gli3 cko mutants (9/16). Furthermore, the cerebellum was well developed, appeared to have a normal cytoarchitecture and had begun to foliate, although the foliation pattern was abnormal (Fig. 1C,G; Fig. 3O-Q; see Fig. S2K,L in the supplementary material). When Gli3 function was left intact until E11.5 (Nes-Gli3 cko mutants), all the morphological defects in the cerebellum, isthmus and tectum were rescued at E18.5 (Fig. 1A,D,E,H), but the size of the isthmus and of the entire tectum was significantly increased (Fig. 1A,D,E,H,L; see Fig. S2M,N in the supplementary material).
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Gli3 is required to regulate mes and r1 growth
To assess when the mes/r1 phenotypes arise in Gli3 mutants, we
analyzed E10.5 (Gli3-/- and En1-Gli3 cko) and
E12.5 (all mutants) embryos. We observed severe morphological defects in the
mes/r1 of Gli3-/- and En1-Gli3 cko mutant
embryos, which in general were more pronounced in Gli3-/-
mutants (Fig. 1M-O and data not
shown): (1) the mesencephalic ventricle was expanded; (2) dorsal posterior
mes, isthmus and r1 were not morphologically distinct from each other and the
isthmic flexure was less prominent; (3) the thickness of the ventricular zone
of the posterior dorsal mes, isthmus and r1 region was increased.
The increased growth of the mes/r1 in Gli3 mutant embryos could be
caused by a decrease in cell death or an increase in proliferation. A previous
whole-mount analysis suggested that cell death is decreased in the mes/r1 at
E8.5 and in the mes dorsal midline at E9.5 in Gli3-/-
mutants (Aoto et al., 2002). To
investigate whether decreased cell death could underlie the expansion of
dorsal mes/r1 in Gli3 mutant embryos, we analyzed the number of
cleaved caspase-3-positive cells in the mes/r1 of E9.5 and E10.5
En1-Gli3 cko mutants. There was a trend toward decreased cell death
in En1-Gli3 cko mutants as compared with WT, but only in the E10.5
dorsal mes/r1 (see Fig. S3D,E in the supplementary material). However, because
these results indicate that cell death is not dramatically reduced in the
absence of Gli3, reduced cell death is unlikely to be the sole cause
of mes/r1 overgrowth in the mutants.
To address whether increased proliferation contributes to the increase in
mes/r1 size, we analyzed ventral and dorsal mes/r1 of WT and cko mutant
embryos for BrdU incorporation (1-hour pulse, S phase of the cell cycle) or
for expression of phosphohistone H3 (PH3) (G2-M phase of the cell cycle).
Quantitative assessment of proliferating cells indicated a slight increase in
proliferation in the dorsal mes/r1 of En1-Gli3 cko mutants at E9.5
and E10.5 and in the dorsal mes of E12.5 Nes-Gli3 cko mutants as
compared with WT (see Fig. S3A-C in the supplementary material). Importantly,
in the ventral mes/r1, proliferation appeared to be similar in cko mutants and
WT, consistent with the lack of a ventral phenotype at E18.5. Since an
increased proliferation rate could be caused by a shortening of cell cycle
length or by a delay in differentiation, we analyzed the cell cycle exit rate
in E12.5 WT and Nes-Gli3 cko mutants
(Chenn and Walsh, 2002). We
quantified the percentage of differentiating cells (BrdU+
Ki67- cells/BrdU+ cells) 24 hours after BrdU
administration and did not observe an obvious changes between WT and mutant in
ventral or dorsal mes or r1 (see Fig. S3F in the supplementary material).
Thus, the increase in proliferation is not due to a decreased ability of cells
to leave the cell cycle. In summary, these data demonstrate that Gli3
is an important regulator of the growth of dorsal r1 (up to E11.0) and mes
(beyond E11.0), modulating both cell proliferation (attenuation) and cell
death (augmentation).
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Despite the abnormal En1 and Otx2 expression in the posterior tectum, this region was not transformed into SC in the mutants, as the AP extent of the Otx2 expression domain in the superficial layers of the SC in Gli3-/- and En1-Gli3 cko mutants was comparable to WT (Fig. 2F,G). Furthermore, neurogranin, which is expressed in differentiated cells in the lateral IC at E18.5 and in the entire IC at P16 (Fig. 2I,L), was found to be expressed in the lateral tectum of E18.5 Gli3-/- mutants. The neurogranin expression domain, however, was severely reduced and shifted anteriorly (3/3) (Fig. 2J). In P16 En1-Gli3 cko mutants, neurogranin immunostaining revealed a more elongated IC domain than in WT (Fig. 2L,M). By contrast, the shape of the IC in P16 Nes-Gli3 cko mutants was comparable to WT, even though the size of the IC was increased (Fig. 2L,N). These data indicate that Gli3 is required for proper establishment of the IC before E9.0, regulates normal IC morphology between E9.0 and E11.0 and growth beyond E11.0. By contrast, Gli3 is not required for establishing the SC, although it plays a prolonged role in controlling normal SC growth.
Gli3 regulates proper establishment of the isthmus and cerebellum before E9.0 and cerebellar foliation between E9.0 and E11.0
To study the cellular phenotype of the isthmus-cerebellum-like region in
Gli3-/- mutants, we analyzed markers for cerebellar and
isthmic cell types. In the E18.5/P0 WT cerebellum, Purkinje cells are
organized in a layer underlying Math1 (Atoh1)-positive granule cell precursors
in the external granule cell layer (Fig.
3A,C,F,I). At this stage, all Purkinje cells express the retinoic
acid receptor-related orphan receptor alpha (ROR
), while a large subset
expresses calbindin and inositol 1,4,5-trisphosphate receptor 1 (IP3R1; Itpr1
- Mouse Genome Informatics) (Fig.
3F,I and data not shown). Pax2 is expressed throughout the isthmus
and deeper cerebellum where it marks (among other cell types) a subset of
interneuron precursors (Fig.
3L) (Maricich and Herrup,
1999).
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- and IP3R1-positive Purkinje cells, indicating that
this region had some characteristics of the cerebellum rather than of the
isthmus (Fig. 3G,J,M and data
not shown). The organization of the posterior isthmus-cerebellum-like region
had some similarities to WT cerebellum, with a relatively normal distribution
of Pax2-positive cells and ROR- and IP3R1-positive Purkinje cells
underlying a Math1-positive external granule cell layer
(Fig. 3B,D,G,J,M and data not
shown). The Purkinje cell layer, however, was thicker than normal and did not
consistently extend into the most posterior area. In addition, Purkinje cell
axons that form bundles in WT were highly disorganized in
Gli3-/- mutants (Fig.
3F,G and data not shown). This disorganization of Purkinje cells
was equally severe in more-lateral regions of the cerebellum (data not shown).
These results indicate that the anterior isthmic-cerebellar region is not
properly specified in Gli3-/- mutants and the posterior
isthmus-cerebellum area is not organized into a normal cerebellar
cytoarchitecture. In P0 En1-Gli3 cko mutants, the defects in cerebellar cytoarchitecture were partially rescued, based on marker analysis (Fig. 3E,H,K,N and data not shown). Purkinje cell axons projected aberrantly into the posterior isthmus and some Purkinje cells formed clusters in the anterior isthmic region (Fig. 3H,K), suggesting that the isthmus-cerebellum boundary was not properly established in En1-Gli3 cko mutants. Furthermore, at E18.5 and all the postnatal stages analyzed (P2, 5, 8, 16 and 30), the cerebellar foliation pattern was clearly abnormal and varied between mutants (Fig. 3O-Q; see Fig. S2K,L in the supplementary material). Interestingly, these defects in foliation were not associated with major changes in AP gene expression domains, as the expression of Otx2 (posterior region) and of Runx1 (central region) in the external granule cell layer were comparable to WT (data not shown). Consistent with the histological analysis, the cytoarchitecture, morphology and foliation pattern of E18.5 and postnatal Nes-Gli3 cko cerebella were similar to those of WT (Fig. 1A,D and data not shown). In conclusion, Gli3 is required primarily before E9.0 for the proper specification of the isthmus and cerebellum, including the formation of normal cerebellar cytoarchitecture, and between E9.0 and E11.0 for establishing the stereotypic cerebellar foliation pattern.
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The unaltered DV gene expression domains in Gli3 mutants indicate
that there is no ectopic GliA-mediated Shh signaling activity in the dorsal
mes/r1 in the absence of Gli3. To definitively demonstrate that
GliA-mediated Shh signaling does not contribute to the patterning and growth
defects in Gli3 mutants, we generated double cko mutants for
Smo and Gli3 (En1-Gli3;Smo cko), as removal of
Smo results in the absence of GliA activity. Indeed, we found that
E12.5 and P0 En1-Gli3;Smo cko mutant embryos had a tectal and isthmus
phenotype very similar to that of En1-Gli3 cko mutants
(Fig. 4K-M; compare
Fig. 4J with
Fig. 1C). The morphology and
overgrowth of r1 in En1-Gli3;Smo cko mutants was also similar to that
shown by En1-Gli3 cko mutants at E12.5. At P0, the overall size of
the cerebellum and thickness of the external granule cell layer, however, were
reduced (Fig. 4J and data not
shown), resembling the En1-Smo cko phenotype that results from
severely decreased granule cell precursor proliferation after E16.5
(Blaess et al., 2006;
Corrales et al., 2006). This
indicates that the overgrowth in the En1-Gli3;Smo cko cerebellar
anlage is initially caused by loss of Gli3R (before E16.5), whereas after
E16.5 the loss of Gli1/2A-mediated Shh signaling downstream of Smo results in
reduced cerebellum growth owing to decreased granule cell precursor
proliferation. In summary, these data demonstrate that the initial phenotypes
in the dorsal mes/r1 of Gli3 mutants result from a loss of Gli3R
activity, rather than from ectopic GliA-mediated Shh signaling.
Gli3 is not required for roof plate induction in the mes/r1
Since defects in the roof plate contribute to the telencephalic phenotype
in Gli3-/- mutants
(Grove et al., 1998;
Theil et al., 2002), we
assessed whether alterations in signaling from the mes/r1 roof plate cause the
dorsal mes/r1 phenotypes of Gli3 mutants. The expression of two
secreted factors involved in the organizing function of the roof plate,
Wnt1 and Gdf7, was maintained in the roof plate of
Gli3-/- and En1-Gli3 cko embryos at E9.5 and
E10.5 (Fig. 5A-F and data not
shown). In addition, Msx1, which is a downstream target of BMP
signaling (Alder et al., 1999;
Bei and Maas, 1998), was
present in the roof plate in Gli3-/- and En1-Gli3
cko mutants (Fig. 5G,H and data
not shown). Finally, expression of Axin2, a target of Wnt signaling
(Jho et al., 2002), was not
grossly altered in E10.5 Gli3-/- or En1-Gli3 cko
mutant embryos (Fig. 5I,J and
data not shown). In summary, Gli3 does not appear to play a major
role in establishing or maintaining the mes roof plate.
Gli3R is required to downregulate Fgf in dorsal r1
Since changes in DV patterning or roof plate signaling do not seem to
account for the dorsal mes/r1 phenotype in Gli3 mutants, we next
examined whether alterations in the Fgf8 signaling pathway, which is a primary
regulator of AP patterning, could contribute to the dorsal mes/r1 defects in
Gli3 mutants. Based on whole-mount analysis, it has been reported
that Fgf8 expression is expanded in E9.5 and E10.5
Gli3-/- mutants, whereas the Fgf8 domain is
reduced when Shh signaling is decreased and Gli3R is increased
(Aoto et al., 2002;
Blaess et al., 2006). We first
analyzed Fgf8 expression in spatial and temporal detail on sagittal
sections of Gli3 mutant embryos. Indeed, we observed ectopic
Fgf8 expression, but only in medial r1 in both E9.5 and E10.5
Gli3-/- (Fig.
6G-I and data not shown) and En1-Gli3 cko (data not
shown) mutants. By E12.5, shortly before the normal termination of
Fgf8 expression, ectopic Fgf8 expression was restricted to
the posterior-most part of medial r1 in Gli3-/- (data not
shown) and En1-Gli3 cko (Fig.
6J,K) mutants. Interestingly, this region corresponds to the
rhombic lip in the WT and normally expresses Wnt1 and Math1
(Fig. 6A,B,D,E and data not
shown). Furthermore, Fgf17, which also plays a role in AP patterning
of the mes/r1 and is expressed in a broader domain than Fgf8 in the
WT (Xu et al., 2000), and
sprouty 1 (Spry1), a direct target of Fgf8 signaling
(Liu et al., 2003), were
expanded posteriorly in both Gli3 mutants, but were not altered in
the mes (Fig. 6M-Q,S-W). To
address whether Gli3R rather than Shh signaling is required for Fgf8
suppression in r1, we analyzed E12.5 En1-Gli3;Smo cko mutants and
found that similar to En1-Gli3 cko mutants, Fgf8, Fgf17 and
Spry1 were expressed ectopically in dorsal-posterior r1 (data not
shown).
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Gli3 mediates isthmic and cerebellar patterning through regulation of Fgf8 expression
To test whether the expansion of Fgf expression in r1 is responsible for
any of the dorsal defects in Gli3 mutants, we removed one copy of
Fgf8 in Gli3-/- mutants. Histological analysis of
Gli3-/-;Fgf8+/- mutants (n=4) at E18.5
or P0 showed a striking partial rescue of the defects seen in the
isthmus-cerebellum-like region, but not tectum, of Gli3-/-
mutants (Fig. 7; see Fig. S4 in
the supplementary material). Unlike any of the Gli3-/-
littermates, the cerebellum of all
Gli3-/-;Fgf8+/- mutants had begun to foliate at
P0 and the external granule cell layer extended along the AP length of the
medial cerebellum (Fig. 7A,E;
for a direct comparison of littermates see Fig. S4A-H in the supplementary
material). In addition, the isthmus appeared to have formed, based on
morphology and the expression of Pax2 (Fig.
7A,H; see Fig. S4A-H in the supplementary material). Marker
analysis further revealed a more normal organization of Purkinje cells,
although cell clusters remained in and near the isthmus region, showing that a
separation of isthmus and cerebellum was not fully established
(Fig. 7F,G; see Fig. S4I,J in
the supplementary material).
Although the isthmus-cerebellar phenotype was partially rescued in the Gli3-/-;Fgf8+/- mutants as compared with Gli3-/- single mutants, the tectal phenotype was similar to Gli3-/- single mutants. The tectum was enlarged, the IC was not clearly distinguishable and the range of morphological abnormalities was comparable to that of Gli3-/- mutants (Fig. 7A; Fig. 1B; see Fig. S2A,C,E,G,I and Fig. S4 in the supplementary material). Furthermore, En1, Otx2 and neurogranin were abnormally expressed in the posterior tectum, similar to in Gli3-/- mutants (Fig. 7B-D; Fig. 2A,B,E,F). These findings provide genetic evidence that a major role of Gli3R in regulating the organization of cerebellar and isthmic cytoarchitecture is to localize Fgf8 expression to dorsal-medial r1.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Gli3R is required to restrict Fgf8 expression to the isthmus
We and others showed previously that Fgf8 expression in the
isthmic organizer is reduced when Gli3R is upregulated (Shh/Smo
mutants) and increased when Gli3 is absent
(Gli3-/- mutants) (Aoto
et al., 2002; Blaess et al.,
2006). We built on these findings by showing that complete absence
of Gli3 as well as inactivation of Gli3 at E9.0
(En1-Gli3 cko mutants) result in an expanded expression domain of not
only Fgf8, but also Fgf17 and the Fgf8 downstream target
Spry1. Furthermore, the expansion occurs only in the dorsal-medial
region of r1 and, interestingly, this region includes the rhombic lip region.
Moreover, the ectopic Fgf expression in r1 in the absence of Gli3 is
not diminished when Shh signaling is abolished (En1-Gli3;Smo cko
mutants). Thus, Gli3R and not Shh activator signaling is required to restrict
Fgf8/Fgf17 expression to the isthmus. These results raise the
question whether Gli3 might regulate Fgf8 expression directly.
Sequence analysis of the known Fgf8 regulatory elements
(Beermann et al., 2006),
however, did not reveal any canonical Gli binding sites
(Vokes et al., 2007) in these
conserved regions (S.B. and J. Hastings, unpublished).
|
). Although there is evidence that the transcription factors
Otx2 and Grg4 (Tle4 - Mouse Genome Informatics) are
normally involved in repression of Fgf8 in the mes, it was unclear how Fgf8
becomes downregulated in dorsal r1 (Ye et
al., 2001). Our study of Gli3 mutants indicates a role
for Gli3R in the initial downregulation of Fgf8 in r1, as well as a
continued requirement that extends beyond E9.0 in maintaining the dorsal
restriction of Fgf8 to the isthmus. Alternatively, Gli3 might be
required to maintain dorsal-medial r1 identity, and the expansion of
Fgf8 expression is a secondary consequence of a mixed r1/isthmus
identity. Interestingly, a similar interaction between Shh, Gli3 and Fgf8
exists in the developing telencephalon
(Gutin et al., 2006;
Kuschel et al., 2003;
Ohkubo et al., 2002),
suggesting that the interplay between these two signaling pathways constitutes
a more general mechanism in the patterning of three-dimensional brain
structures.
Regulation of cerebellum and isthmus development by Gli3R is dependent on restricting Fgf8 to the isthmus
Based on Fgf8 loss- and gain-of-function studies, the observed ectopic
expression of Fgf8 in dorsal r1 of Gli3 mutants could affect
both tectum and cerebellum development
(Zervas et al., 2005). To test
this possibility, we analyzed Gli3-/- mutants in which one
copy of Fgf8 was removed. Strikingly, we found that whereas the
isthmic-cerebellar region phenotype is partially rescued in
Gli3-/-;Fgf8+/- mutants, the defects in tectal
growth and IC patterning were largely not attenuated. The lack of influence of
Fgf8 on mes development is likely to be because an insufficient level of Fgf8
signaling is attained in the mes, consistent with the observation that
Spry1 is not obviously upregulated in the mes of Gli3
mutants.
|
|
Attenuation of mes growth by Gli3R
One striking phenotype in the Gli3-null and cko mutants is the
extensive overgrowth of tectal tissue. It has previously been shown that
ectopic activation of the Shh pathway by misexpression of either Shh or GliA
results in a pronounced overgrowth of the spinal cord, mes/r1 and postnatal
cerebellum in chick and mouse (Cayuso et
al., 2006; Corrales et al.,
2006; Dahmane and
Ruiz-i-Altaba, 1999; Rowitch
et al., 1999). The increased size of the tectum that we documented
in Gli3 and Gli3/Smo mutants, however, demonstrates that
removing Gli3R, without an associated ectopic activation of Gli activator
function, can cause increased proliferation and overgrowth. Furthermore, Gli3R
is normally required beyond E11.5 to attenuate growth of the dorsal mes.
Our present results taken together with previous studies
(Blaess et al., 2006;
Britto et al., 2002;
Ishibashi and McMahon, 2002;
Corrales et al., 2006) suggest
several mechanisms by which Gli transcription factors regulate tissue growth.
At high levels of signaling, Shh can simply increase proliferation by
upregulating GliA (primarily Gli2) such as in cerebellar granule cell
precursors (Corrales et al.,
2006), and/or by downregulating Gli3R such as in the ventral
midbrain (Blaess et al.,
2006). At low levels of Shh signaling, such as in the dorsal mes,
GliA is not induced, and Shh increases overall growth only by downregulating
Gli3R levels which leads to increased proliferation and cell survival
(summarized in Fig. 8B). The
latter reveals that other signaling pathways must stimulate proliferation of
dorsal mes progenitors and a certain level of Gli3R is required only to
attenuate growth.
A possible candidate to regulate proliferation in the dorsal mes is Wnt1,
as it can induce overgrowth of the posterior mes
(Panhuysen et al., 2004) and
recent studies have provided evidence that Gli3R inhibits canonical Wnt
signaling in several different tissues and cell types
(Alvarez-Medina et al., 2008;
Ulloa et al., 2007). However,
our analysis of Axin2 expression (a readout for canonical Wnt
signaling) in the mes of Gli3 mutants provided no evidence that
Gli3 is required in the dorsal mes to antagonize Wnt signaling.
Furthermore, our preliminary analysis of
Gli3-/-;Wnt1sw/sw double mutants revealed that
the size of the SC is comparable to that of Gli3-/-
mutants, although the IC (and medial cerebellum) is lost in both
Wnt1sw/sw and
Gli3-/-;Wnt1sw/sw mutants (S.B., unpublished).
It is possible, however, that additional members of the Wnt family stimulate
dorsal mes growth and that their action is normally attenuated by Gli3R.
In summary, we have found that the precise regulation of Gli3R levels is crucial to establish the intricate structures of the mature tectum and cerebellum. Our data provide insight into how Gli3R coordinates the function of two distinct organizer molecules in the mes/r1 by modulating their expression or downstream signaling. It will be interesting to investigate whether Gli3R plays similar complex roles in other tissues that undergo organizer-dependent three-dimensional patterning and growth.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/12/2093/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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