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First published online May 16, 2007
doi: 10.1242/10.1242/dev.000729
Department of Cell and Developmental Biology, Vanderbilt University Medical Center, 4114 MRB III, Nashville, TN 37232, USA.
* Author for correspondence (e-mail: chin.chiang{at}vanderbilt.edu)
Accepted 1 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Sonic hedgehog, Lipid modification, Telencephalon, Cholesterol, Patterning, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Dale et al.,
1997; Fuccillo et al.,
2006; Jessell,
2000; Sur and Rubenstein,
2005). In mice lacking Shh, most ventral neuronal cell types are
lost along the rostral-caudal axis of the neural tube, whereas dorsal
structures are expanded ventrally (Chiang
et al., 1996; Rallu et al.,
2002). It is thought that neurons generated in progressively more
ventral regions in the spinal cord require correspondingly higher levels of
Shh for their induction (Briscoe and
Ericson, 2001; Jessell,
2000). In the basal ganglion of the telencephalon, the medial
ganglionic eminence (MGE) and lateral ganglionic eminence (LGE), which give
rise to the globus pallidus and striatum, respectively, are generated
sequentially in response to Hedgehog (Hh) signaling
(Ericson et al., 1995;
Kohtz et al., 1998;
Shimamura and Rubenstein,
1997).
Although Shh can elicit diverse cell fates in the neural tube, much less is
known about the factors that regulate Shh range and activity gradient. Active
Shh is dually lipid-modified (ShhNp), with palmitic acid at its N-terminus and
cholesterol at its C-terminus (Mann and
Beachy, 2004). The hydrophobic nature of lipids indicates that
there might be a dedicated release mechanism for Shh. Disp1, a multi-pass
transmembrane protein, appears to fulfil this mechanistic requirement
(Caspary et al., 2002;
Kawakami et al., 2002;
Ma et al., 2002), as its
function is necessary only for lipidated Shh secretion
(Li et al., 2006;
Tian et al., 2005).
Lipid-modified Shh has also been shown to form a large multimeric complex in
vitro (Chen et al., 2004a;
Zeng et al., 2001). It has
been suggested that the lipid moieties are embedded in the core of these
complexes, analogous to micelles, thus facilitating Shh interaction with - and
its spreading across - the extracellular matrix
(Zeng et al., 2001). The
biological function of lipid moieties in regulating Shh function has been
investigated recently. Misexpression studies in rats indicated that
palmitoylation is required for Shh to induce ectopic ventral cell fates in the
dorsal telencephalon (Kohtz et al.,
2001), suggesting that palmitoylation is required for Hh activity.
Indeed, mice deficient in an enzyme that catalyzes palmitoylation, or carrying
a variant of Shh that is incapable of being palmitoylated, showed
developmental defects similar to those of loss of Shh function
(Chen et al., 2004a).
In contrast to palmitoylation, lack of cholesterol modification (ShhN) does
not appear to significantly affect the intrinsic potency of Shh. It has been
shown that ShhN isolated from tissue culture cells can induce ectopic ventral
cell types in neural explants with comparable or higher efficiency than ShhNp
at similar concentration (Feng et al.,
2004). Instead, the cholesterol moiety appears to affect the range
of Hh spreading in the target field. Recent mosaic studies with
Drosophila wing imaginal discs showed that Hh lacking cholesterol
(Hh-N) has an extended range of spreading and for inducing ectopic
low-threshold Hh target genes, although the expression of high-threshold
target genes near the source was reduced
(Callejo et al., 2006;
Dawber et al., 2005;
Gallet et al., 2006). However,
another report indicated that wing disc clones that expressed Hh-N were unable
to elicit low-threshold Hh target genes at a distance
(Gallet et al., 2006). These
differences might be associated with different levels of Shh that are
expressed at the sites of synthesis
(Guerrero and Chiang,
2006).
Previous efforts to understand the role of cholesterol modification
concluded that the cholesterol moiety is required for long-range spreading and
signaling in the mouse limb buds (Lewis et
al., 2001). Accordingly, it has been proposed that cholesterol
modification is required for the formation of Shh multimers involved in
long-range signaling and proper vertebrate forebrain patterning
(Feng et al., 2004). However,
a more recent study suggested that ShhN retained some paracrine activity
because it was capable of inducing several ventral neuronal cell types in the
spinal cord (Tian et al.,
2005). Given this controversy, the role of the cholesterol moiety
in Shh function has been re-assessed recently in the limb bud
(Li et al., 2006). That study
found that cholesterol modification is required to restrict the spread of Shh
across the anteroposterior axis of the limb bud, similar to Hh-N function in
Drosophila. The extended range of ShhN movement across the
anteroposterior axis of the limb bud also resulted in an appreciable reduction
of local ShhN levels, suggesting that the cholesterol moiety not only
regulates the range, but also the shape of the Shh morphogen gradient.
In this study, we utilized mice exclusively expressing the ShhN form of Shh to address the function of the cholesterol moiety in patterning the spinal cord and telencephalon. We found that dorsoventral patterning of the telencephalon in embryos lacking cholesterol modification of Shh was severely compromised, as indicated by the lack of MGE progenitors and the appearance of widespread ectopic LGE progenitors in the dorsal telencephalon. By contrast, patterning of the ventral spinal cord was largely unaffected and no ectopic expression of ventral cell fates at dorsal sites was observed in ShhN/- embryos. These findings suggest that patterning of the telencephalon is highly sensitive to alterations in Shh spreading behavior.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The resulting mice containing the Shhflox (unrecombined)
allele were maintained as heterozygotes, because homozygotes die soon after
birth (Li et al., 2006). The
Shhflox allele has reduced capacity to generate
lipid-modified Shh (ShhNp) owing to interference of the loxP site with the Shh
autocatalytic processing reaction (Li et
al., 2006) (data not shown), thus it should be considered
hypomorphic. Embryos uniquely expressing Shh without cholesterol modification
were generated by mating Shhflox/+ animals with the
Shh+/-;Sox2-Cre deleter strain as previously described
(Li et al., 2006).
ShhN/+;Disp1-/- embryos were obtained from
crosses between Shhflox/+;Disp1+/- and
Sox2-Cre;Disp1+/- mice. Disp1+/- mice
have been described (Ma et al.,
2002). Sox2-Cre mice
(Hayashi et al., 2002) and
Ptch1-lacZ mice (Goodrich et al.,
1997) were obtained from the Jackson Laboratory. All control
embryos were either Shh+/+ or Shh+/-;
we have not observed any differences in the development or Shh staining
patterns of these embryos (see Fig. S3 in the supplementary material).
Immunohistochemistry and western blot analysis
Immunohistochemistry analyses were performed on tissue sections collected
from OCT- or paraffin-embedded embryos as previously described
(Li et al., 2006). The primary
antibodies used were rabbit anti-Foxa2 (Li
et al., 2007) (1:100), mouse anti-Nkx2.2 (DSHB, 1:2), mouse
anti-Isl1/2 (DSHB, 1:10), sheep anti-Chx10 (Exalpha Biologicals, 1:50), mouse
anti-En1 (DSHB, 1:5), mouse anti-Pax7 (DSHB, 1:2), mouse anti-Pax6, (DSHB,
1:1), mouse anti-Nkx2.1 (Neomarkers, 1:100), rabbit anti-Nkx6.1 (1:3000, gift
of Christer Betsholz, Karolinska Institute, Stockholm, Sweden), rabbit
anti-Olig2 (1:3000, gift of Hirohide Takebayashi, Kyoto University, Yoshida,
Japan), mouse anti-Mash1 (1:100, gift of Jane Johnson, University of Texas
Southwestern Medical Center, Dallas, TX, USA), rabbit anti-Gsh2 (1:500, gift
of Kenneth Campbell, University of Cincinnati, Cincinnati, OH, USA), mouse
anti-Tuj1 (Sigma, 1:400), goat anti-Shh (Santa Cruz Biotechnology, 1:1000).
For western analysis, protein lysate samples (200 µg each), collected from
E15.5 whole brains, were resolved on 6% SDS-polyacrylamide gels. Gli3-190 and
Gli3R species were detected using a Gli3 N-terminal-specific antibody as
previously described (Litingtung et al.,
2002).
Analysis of cell proliferation and apoptosis
5-Bromodeoxyuridine (BrdU) in vivo labeling and TUNEL analysis were
performed as previously described
(Litingtung et al., 1998).
Proliferative and apoptotic cell countings were carried out with at least five
sections from three different embryos for each genotype. To quantify BrdU- or
TUNEL-positive cells, coronal sections of the telencephalic neuroepithelium
were subdivided into three regions: ventral (v), dorsal (d) and dorsal midline
(dm). v is defined as the region of neuroepithelial cells ventral to the
dorsal edge of the morphologically distinct LGE (see
Fig. 2, arrowhead). The dm
region consists of 200 neuroepithelial cells situated at the center of the
dorsal midline and its immediate neighbors. The d region is located between
regions v and dm. The percentage of BrdU-positive cells was determined by
counting these in segments of 200 cells within the designated region of the
telencephalon and dividing by the total number of cells in the segment. All
sections were counterstained with the nuclear dye DAPI to highlight the total
number of cells. At least five segments in each region were counted to
generate data for statistical comparison.
X-Gal staining and transcript detection
X-Gal staining was performed according to standard protocols
(Hogan et al., 1994).
Whole-mount and section in situ hybridizations were performed as described
(Li et al., 2004). The
following cDNAs were used as templates for synthesizing digoxygenin-labeled
riboprobes: Shh (Li et al.,
2006), Ptch1
(Goodrich et al., 1996),
Gli3 (Li et al.,
2004) and Nkx2.1
(Lazzaro et al., 1991).
Statistics
To assess differences among groups, statistical analyses were performed
using a one-way analysis of variance (ANOVA) with Excel (Microsoft) and
significance accepted at P<0.05. Results are presented as
mean±s.e.m.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We next determined whether the capacity for neural progenitor cells to proceed with differentiation is altered in ShhN/- mutant telencephalon. We stained ShhN/- and control sections with an antibody against neuronal class III tubulin (Tuj1; Tubb3 - Mouse Genome Informatics) that marks all differentiated neurons. At E11.5, Tuj1-positive cells normally consisted of a thick layer of differentiated neurons within the subventricular zone and mantle zone of MGE and LGE (Fig. 2O). However, in ShhN/- mutants, we observed significant reduction in the population of differentiating neurons within the presumptive LGE domain (Fig. 2T). There was no obvious difference in differentiation in the cortex and dorsal midline region between control and ShhN/- embryos at this stage (Fig. 2P,Q,U,V). Similarly, Isl1, a ventral-specific differentiation marker, also showed significant reduction in the differentiation capacity of ShhN/- ventral neural epithelium (Fig. 2R,S,W,X). Collectively, these observations suggest that increased apoptosis and reduced neuronal differentiation contribute to the defective development of LGE in ShhN/- mutants.
In addition to basal ganglions, dorsal midline derivatives such as the
secretory epithelial cells of the choroid plexus and hippocampus primodium
were also affected in ShhN/-
(Fig. 1H,J). The rapid
invagination and remodeling of the dorsal midline neuroepithelium is normally
accompanied by reduced proliferation and increased cell death
(Furuta et al., 1997;
Nagai et al., 2000). Although
cell death still occurred in the ShhN/- dorsal roof
(Fig. 2C,F; 41±5.7
versus 44±6.9, P=0.48), we observed a near 100% increase in
cell proliferation in the roof and medial wall of the dorsal midline
(Fig. 2J,M, arrowheads;
34.4±1 versus 72±3, P<0.001), suggesting that ShhN
might have long-range effects on dorsal midline development (see below). Taken
together, these results indicate that the cholesterol moiety of Shh is
required for normal development of both dorsal and ventral telencephalic
structures.
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).
At E12.5, Nkx2.1 was uniformly expressed in the MGE of the control embryo,
whereas no Nkx2.1 expression could be detected in the
ShhN/- mutant telencephalon, indicating severe loss of MGE
identity (Fig. 3A,B).
Mash1, which encodes a neural basic helix-loop-helix transcription
factor, is expressed in the pan-ventral telencephalic region, including the
MGE and LGE (Casarosa et al.,
1999; Horton et al.,
1999; Lo et al.,
1991). Mash1 expression in these regions is regulated by Hh
signaling and marks a transient population of committed neural precursors
before differentiation (Fuccillo et al.,
2004). Although Mash1 expression was present in both MGE and LGE
of control embryos, Mash1-positive cells encompassed a relatively large
ventral portion of the ShhN/- mutant telencephalon
(Fig. 3E,F), indicating that
LGE progenitor cells were present despite the lack of development of a
morphologically distinct LGE structure. Pax6 expression is normally confined
to the dorsal cortical region abutting the LGE boundary
(Fig. 3I). We observed
prominent expansion of Pax6 expression in the ventral neuroepithelium, with
reduced levels in the presumptive LGE domain
(Fig. 3J, arrowheads). The
expanded Pax6 expression domain was observed in the Shh-/-
mutant telencephalon, which often collapsed at this stage with no expression
of ventral markers such as Mash1 and Nkx2.1
(Fig. 3D,H,L). Although these
results point to a requirement for the cholesterol moiety of Shh in patterning
the ventral telencephalon, the possibility remains that the expression level
of the ShhN allele is not as high as the wild-type allele when it is
expressed alone in ShhN/- embryos, thus generating
haploinsufficiency. Because it is not possible to generate ShhN
homozygous mutants (see Materials and methods), we compared the patterning
capacity of the Shhflox allele, in which the ShhN
allele is generated upon Cre-mediated deletion of the sequence encoding the
C-terminal autoprocessing domain. With the exception of the cholesterol
moiety, the proteins generated from these two alleles are the same. However,
the Shhflox allele is hypomorphic in that it generates a
lower level of bioactive lipid-modified ShhNp owing to loxP sequence
interference with autoprocessing of the Shh precursor
(Li et al., 2006) (data not
shown). Thus, Shhflox/- embryos died soon after birth with
multiple defects, including cleft secondary palate and distorted eye formation
(see Fig. S1A-E in the supplementary material), characteristics of impaired
Shh signaling (Rice et al.,
2004; Zhang and Yang,
2001). However, dorsoventral patterning of the telencephalon was
maintained in hypomorphic Shhflox/- mutants, although a
reduction of the LGE domain was evident
(Fig. 3C,G,K). Therefore, the
severe patterning defect in ShhN/- is primarily attributed
to failure of cholesterol modification rather than haploinsufficiency, which
is also consistent with the ectopic activation of Shh pathway activity
observed in the dorsal telencephalon (see below).
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The emergence of ectopic LGE marker expression and defective midline dorsal
structures at late gestation in ShhN/- are consistent with
the notion that the dorsal telencephalon might be exposed to low levels of
ectopic ShhN signal. Indeed, we detected low but consistent Shh pathway
activation in the dorsal telencephalon of ShhN/- mutants
as indicated by sensitive Ptch1-lacZ reporter expression
(Fig. 5B), whereas no dorsal
Shh signaling was found in a comparable region of the control embryo
(Fig. 5A). Because Shh pathway
activation leads to the accumulation of full-length Gli3 (Gli3-190) through
the inhibition of Gli3 repressor (Gli3R) formation
(Litingtung et al., 2002;
Wang et al., 2000), we
determined the relative amount of Gli3R to Gli3-190 as another readout of Shh
signaling. Consistent with widespread Ptch1 reporter expression, we observed
as much as a 50% reduction in the Gli3R/Gli3-190 ratio in E15.5 whole-brain
extracts of ShhN/- mutants as compared with controls
(Fig. 5C,D). Ectopic Shh
signaling is not the result of aberrant ectopic Shh expression, as we did not
observe Shh-expressing cells or their descendents in the dorsal telencephalon
using the Shh-Cre transgenic line that we described previously
(Li et al., 2006) (data not
shown). Thus, it appears that the cumulative effect of long-range ectopic ShhN
signaling resulted in the induction of ventral marker expression in the dorsal
neural progenitor population.
The cholesterol moiety is required for progenitor cell expansion but not subtype specification in the spinal cord
In order to determine whether specification of ventral cell types was
similarly compromised in ShhN/- mutant spinal cord, as in
the telencephalon, we analyzed the expression patterns of several homeodomain
transcription factors that identify different cell types including neuronal
progenitors in the spinal cord (Briscoe and
Ericson, 2001; Jessell,
2000). Pax7 is a class II factor that is repressed by Shh and is
expressed in all dorsal progenitor domains, whereas Nkx6.1 (Nkx6-1 - Mouse
Genome Informatics) is a class I factor that is induced by Shh and expressed
in the broad ventral progenitor domains of V2 (p2), V3 (p3) interneurons and
motor neurons (pMN). Similarly, Olig2 and Nkx2.2 (Nkx2-2 - Mouse Genome
Informatics) are class I factors that are selectively expressed in the pMN and
p3 progenitors, respectively. In ShhN/- mutants, all
ventral cell types were generated at defined locations
(Fig. 6), in agreement with
previously reported ShhN function in the spinal cord
(Tian et al., 2005). The
number of floorplate cells labeled by Foxa2 was comparable between control and
ShhN/- embryos (Fig.
6C,D,N). However, there was an overall reduction in the total
number of ventral neuronal progenitor domains and corresponding
Chx10+ (V2, P=0.001), En1+ (V1,
P=0.001) and Isl1+ (MN, P=0.007) neuronal
subtypes that correlated with the reduced neural tube size in
ShhN/- embryos (Fig.
6N,O). Taking into account the difference in neural tube size at
E10.5 (the control spinal cord was about 44% larger than that of
ShhN/-; see Fig. S2J in the supplementary material), the
Nkx6.1+ (p3, pMN and p2) progenitor domains in
ShhN/- were slightly reduced as compared with the control
(Fig. 6M, 35±3% versus
31±3%, P=0.05). Notably, there was an apparent expansion of
the Pax7+ progenitor domain
(Fig. 6M, 49±2% versus
63±2%, P<0.001) and concomitant reduction of p0+p1 (V0 and
V1 progenitors) progenitor domains (Fig.
6M, 16±2% versus 6.2±1%, P<0.001). The
expansion of the Pax7+ domain is not a consequence of maintaining
dorsal tissue mass as a result of reduced neural tube size, as ventral
expansion of dorsal progenitors was still observed at E9.5 when
ShhN/- spinal cord exhibited a size comparable to the
control (see Fig. S2A-H in the supplementary material). These results indicate
that although neuronal subtypes are specified and patterned in
ShhN/- spinal cord, the cholesterol moiety of Shh is,
nevertheless, required for normal expansion of ventral progenitors,
particularly p0+p1 progenitor populations.
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;
Shimamura and Rubenstein,
1997) (Fig. 7A,C).
In ShhN/- embryos, Shh RNA expression in the
dorsal foregut was maintained (Fig.
7E, arrow), but the level of ShhN protein was significantly
reduced (Fig. 7G, arrow).
Accordingly, Shh RNA expression in the overlying neuroepithelium was
almost undetectable (Fig. 7E,G,
arrowheads). By E9.5, Shh expression was completely abolished,
although its expression in other regions of the brain was maintained
(Fig. 7B,F). Similarly, we
failed to detect ShhN protein in ShhN/- ventral
telencephalon, whereas Shh protein was always observed in the midline of
control embryos (Fig. 7D,H). As
expected, Nkx2.1 expression, which overlapped with Shh expression in the
ventral telencephalon, was not observed
(Fig. 7I,M). The expression of
Ptch1, a downstream target of Shh signaling
(Goodrich et al., 1996), was
also severely compromised in this region
(Fig. 7J,N). In contrast to the
telencephalon, Shh transcript and ShhN protein expression in the
floorplate and notochord at E10.5 were clearly detectable, but the ShhN
protein level was significantly reduced
(Fig. 7K,L,O,P). Interestingly,
ShhN protein predominantly localized to the apical membrane of floorplate
cells (arrowheads in Fig. 7P),
whereas ShhNp protein localized to both apical and basolateral membranes. This
is in agreement with a recent study in Drosophila in which Hh-N
accumulated only at the apical membrane
(Callejo et al., 2006).
Intriguingly, we found that ShhNp protein was distributed along the apical
cell membrane of ependymal cells (Fig.
7L, arrows), which line the central canal of the spinal cord. By
contrast, we never observed ependymal localization of ShhN in the mutant
spinal cord. It is possible that this ependymal cell layer represents an
active site where ShhNp is normally concentrated and functions to properly
expand ventral progenitor populations. Thus, the results suggest that ShhN has
a propensity to spread away from its site of synthesis at the expense of its
local concentration, similar to the situation observed in the limb bud
(Li et al., 2006). As Shh
expression in the ventral telencephalon is induced by the underlying
prechordal plate (Ericson et al.,
1995; Shimamura and
Rubenstein, 1997), the ventral patterning defects observed in the
ShhN/- telencephalon are likely to be attributable to the
inability of telencephalic neuroepithelium to establish Shh
expression in response to altered Shh spreading behavior in the prechordal
plate.
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; Kawakami et al.,
2002; Ma et al.,
2002). However, the gross morphology of
Disp1-/-;ShhN/+ embryos largely resembled that
of ShhN/- embryos, with enlarged brain ventricles, closely
positioned frontal nasal process and defective eye formation
(Fig. 8B, data not shown). As
expected, we observed unperturbed dorsoventral neuronal patterning in E10.5
Disp1-/-;ShhN/+ spinal cord, as demonstrated by
well-defined dorsal Pax7 and ventral Nkx6.1 marker expression
(Fig. 8G). Notably, we found an
expanded pool of floorplate cells in
Disp1-/-;ShhN/+ spinal cord that often
co-expressed the V3 interneuron marker Nkx2.2 and the floorplate marker Foxa2
(Fig. 8H-J). This is consistent
with the notion that ShhN protein is capable of inducing floorplate formation
and ShhNp cannot travel beyond the floorplate in the absence of Disp1
function, resulting in an elevated local concentration of Shh in the
floorplate region (Fig. 8F,
note how the robust floorplate Shh staining and the absence of ependymal Shh
staining differ from that of control and ShhN/-
embryos). By contrast, the Disp1-/-;ShhN/+ telencephalon showed patterning defects similar to that of ShhN/- embryos at E12.5, with the absence of MGE neurons and ventral expansion of the dorsal marker Pax6 (Fig. 8C-E), commensurate with the absence of Shh protein in the telencephalon (Fig. 8A). The above results support the notion that there is a more stringent requirement for cholesterol-modified Shh in patterning the ventral telencephalon than the spinal cord.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Enlarged brain ventricles in the ShhN/- mutant
One of the characteristic features in early development of the vertebrate
brain is the appearance of brain ventricles. Generating a uniform and
coordinated outgrowth of neuroepithelium from the neural tube involves
balanced cell proliferation, apoptosis and differentiation. Previous studies
provided evidence that Shh is an essential player to initiate and maintain
ventricle expansion (Britto et al.,
2002). Ablating Shh signaling by removing notochord resulted in
elevated apoptosis and significantly reduced cell proliferation throughout the
neuroepithelium of the developing avian midbrain and forebrain
(Britto et al., 2002). This
growth retardation resulted in a collapse of the telencephalon and
metencephalon ventricles. Furthermore, gradual blockade of Shh signaling by
increasing amounts of cyclopamine led to progressive brain collapse associated
with defective cell proliferation and massive apoptosis
(Cooper et al., 1998).
Shh-/- forebrain development was also severely affected
and it developed into one fused ventricle
(Chiang et al., 1996). In
contrast to reduced or collapsed brain ventricles associated with a reduction
or absence of Shh signaling, ShhN/- embryos showed
enlarged forebrain ventricles, which were more evident at later stages of
development when ectopic activation of Shh signaling was detectable. This
observation suggested that the cholesterol moiety of Shh plays a key role in
maintaining forebrain ventricle expansion by regulating the range of Shh
spreading.
Temporal dependence of ectopic signaling mediated by ShhN
The ability of ShhN to enhance dorsal midline cell proliferation and elicit
ectopic Shh-responsive gene expression in the dorsal telencephalon is
consistent with the capacity of ShhN to spread over a long range. However, the
induction of these genes can only be detected beginning at E13.5, suggesting
that either the dorsal telencephalon is incompetent to receive the ShhN
signal, or the long-range ShhN concentration is below the threshold for target
gene expression at earlier stages. We favor the latter possibility, as
previous neural explant studies showed that the competence to receive Shh
signal in the dorsal telencephalon was diminished by E11.5
(Kohtz et al., 1998).
Interpretation of a morphogen signal by responding cells is achieved by the
signaling intensity, which is conveyed by the concentration of ligand and the
duration of exposure (reviewed by Gurdon
and Bourillot, 2001). Long-lasting residence of ligand-receptor
signaling complex has been shown to lead to prolonged signaling events in
activin-dependent TGF-ß signaling
(Jullien and Gurdon, 2005).
Interestingly, the activated form of smoothened (Smo), a seven-pass
transmembrane receptor that activates downstream effectors of the Shh pathway,
is internalized in a Shh-dependent manner
(Chen et al., 2004b). Recent
time-course analysis suggested that spinal cord progenitor cells could
integrate the level of signaling over time, providing evidence that the
signaling duration is an important parameter, in addition to signaling
strength, to control dorsoventral patterning of the spinal cord
(Stamataki et al., 2005).
Therefore, we speculate that prolonged exposure of the telencephalic
neuroepithelium to ShhN has a cumulative influence on the mediation of the
gradual deposition of activated Smo intracellularly, which might in turn lead
to widespread expression of Shh-responsive genes such as Gsh2 and
Mash1 in ShhN/- telencephalic neuroepithelium at
later stages.
Regulation of long-range spreading of Shh by its cholesterol moiety in the neural tube
Although the mechanism of Shh spreading is not well understood,
lipid-dependent Hh multimers have been proposed to regulate Hh spreading by
interacting with extracellular matrix molecules such as heparan sulfate
proteoglycans (HSPGs) (Callejo et al.,
2006). Furthermore, hedgehog-interacting protein (Hhip), a
membrane glycoprotein, can bind to Shh, thereby inhibiting its spread
(Chuang et al., 2003;
Chuang and McMahon, 1999). A
recent mathematical model for Shh signaling dynamics predicted that the
restrictive effect of these Shh-interacting molecules lowers the diffusion
constant and concentrates the ligand near its secreting source
(Saha and Schaffer, 2006). It
is therefore possible that ShhN protein can escape the synergistic regulatory
mechanisms exerted by Shh-interacting molecules, generating a high diffusion
rate as predicted by the model (Saha and
Schaffer, 2006). Given that ShhN transcript is
undetectable in the ventral telencephalon and that ShhN would have to traverse
across the neural epithelium - a relatively long distance - to activate
Shh-responsive gene expression extending to the dorsal midline, we favor the
idea that ShhN could spread into the subjacent cerebrospinal fluid (CSF) from
its site of synthesis in the neural tube. This proposition is analogous to the
situation in Drosophila, where Hh-N is expressed from peripodial
cells of the wing imaginal disc and is proposed to be secreted into the lumen
to activate low-threshold target genes as it accumulates near the apical
surface (Gallet et al.,
2006).
Diffusive molecules and proteins of the CSF can play crucial roles in
regulating neuroepithelial cell behavior and it has been shown that CSF
promotes neuroepithelial cell survival, proliferation and neurogenesis in
mesencephalic explants (Gato et al.,
2005; Parada et al.,
2005). Recent studies have suggested that the Slit family of
chemorepulsive proteins may be secreted into the CSF to generate a
cilia-dependent concentration gradient necessary for vectorial migration of
neuroblasts in the brain (Sawamoto et al.,
2006). In this context, it is interesting to note that
Shh-expressing Cos cells implanted within the lumen of the midbrain can rescue
midbrain expansion defects generated by extirpation of the notochord,
suggesting that the precise source of Shh signaling activity is not crucial
for normal morphogenesis of early brain ventricles
(Britto et al., 2002).
Furthermore, we found that ventral spinal cord ependymal cells might be an
important site at which ShhNp protein concentrates and/or through which it
travels. Intriguingly, we found by immunostaining that ShhNp protein can be
visualized along the apical side of the innermost ventricular zone
neuroepithelial cells as early as E9.5. Although the significance of apical
surface neuroepithelial expression is not clear, the fact that this pattern is
absent in the ShhN mutant suggests that it might be required for expansion of
ventral progenitor domains. Recent studies in the chick spinal cord have also
reported similar apical surface neuroepithelial expression at the onset of
oligodendrogenesis, suggesting a role of ShhNp in the expansion of
oligodendrocyte precursors (Danesin et
al., 2006). We envision two possible explanations for how Shh is
localized to this region. First, a significant amount of Shh protein could
travel in a planar direction through the innermost neuroepithelial cell layer,
and subsequently spreads laterally into the ventricular zone to establish
different neuronal progenitor identities. Alternatively, it is possible that
ShhNp protein from the floorplate is secreted into the CSF, and ShhNp
receptors that reside on the apical membrane of neuroepithelial cells function
to concentrate and endocytose this ShhNp from the CSF. These two tentative
explanations are not mutually exclusive. Megalin (Lrp2) is a member of the
low-density lipoprotein (LDL) receptor-related protein family. Megalin
function is essential for forebrain formation and its abundant expression at
the apical surface of the CNS neuroepithelium at midgestation is very similar
to ShhNp expression, notably at the apical surface of neuroepithelial cells
(Assemat et al., 2005;
Willnow et al., 1996). A
mechanism has been proposed in which megalin is required to supply sufficient
cholesterol to the rapidly dividing neuroepithelium before neural tube
closure, possibly through the endocytosis of cholesterol-containing
lipoprotein (Herz et al.,
1997). Since it has been shown in tissue culture studies that
megalin could function as an endocytic Shh receptor
(McCarthy et al., 2002), it is
possible that megalin has a direct role in propagating Shh signaling. Another
endocytic receptor, cubilin (Cubn - Mouse Genome Informatics), an EGF-CUB
protein, has a nearly identical apical surface neuroepithelial expression
pattern to megalin (Assemat et al.,
2005). Interestingly, a cubulin-related protein encoded by the
zebrafish scube2 gene has been shown to mediate Shh signaling in
embryos (Hollway et al., 2006;
Woods and Talbot, 2005).
Therefore, it is possible that these two proteins act synergistically in
promoting Shh signaling during mammalian development. It would be interesting
to investigate the functional relationship of Shh, megalin and cubulin in
ependymal cells and the existence of secreted Shh protein in the CSF.
Region-specific effects of ShhN in patterning the neural epithelium
Our study suggests that alteration of Shh spreading behavior has
differential effects in patterning the telencephalon versus spinal cord. This
is most likely to be attributable to regional differences in the generation
and maintenance of Shh expression in the neuroepithelium. In the
spinal cord, the development of ventral progenitor cells is mediated by Shh
secreted from the notochord and floorplate
(Jessell, 2000). Shh
expression in the floorplate is induced and maintained by the notochord, which
is in close contact with the overlying spinal cord for an extended period of
time. In the telencephalon, the development of basal ganglia appears to be
mediated initially by Shh secreted from the prechordal plate (the dorsal
foregut) and then by the ventral neuroepithelium
(Ericson et al., 1995;
Shimamura and Rubenstein,
1997). In contrast to the floorplate, Shh expression in
the ventral telencephalic neuroepithelium becomes quickly independent of the
influence of the underlying prechordal plate. Thus, a reduction in local Shh
concentration in the prechordal plate and notochord, as shown in
ShhN/- embryos, would have more profound effects on
Shh expression and maintenance in the telencephalon than in the
spinal cord.
An unexpected finding is that ShhN exerted no effect on dorsal progenitors,
as there was no evidence of expansion or ectopic ventral progenitor cell fates
in the dorsal region of the spinal cord. This observation is in contrast to
findings in the telencephalon and limb buds
(Li et al., 2006), where
ectopic Shh pathway activation was detected far from the source of ShhN
synthesis. There are at least two, non-mutually exclusive explanations for
this difference. First, temporal responsiveness to ectopic ShhN might be
different between dorsal telencephalon and dorsal spinal cord. Neural explant
studies in rats indicated that E11.5 dorsal telencephalon (
E10.5 in
mouse) remained competent to receive exogenous Shh stimulation
(Kohtz et al., 1998), whereas
E11 (E10 in mouse) spinal cord did not demonstrate such competence
(Charron et al., 2003).
Likewise, the spinal cord might have lost competence to respond to ectopic
ShhN by the time the signal reaches a threshold concentration necessary for
cell fate specification. Alternatively, the concentration threshold required
for ShhN to elicit ectopic patterning effects in the dorsal spinal cord is
higher than for the dorsal telencephalon.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2095/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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.
Assemat, E., Chatelet, F., Chandellier, J., Commo, F., Cases, O., Verroust, P. and Kozyraki, R. (2005). Overlapping expression patterns of the multiligand endocytic receptors cubilin and megalin in the CNS, sensory organs and developing epithelia of the rodent embryo. Gene Expr. Patterns 6,69 -78.[Medline]
Briscoe, J. and Ericson, J. (2001). Specification of neuronal fates in the ventral neural tube. Curr. Opin. Neurobiol. 11,43 -49.[CrossRef][Medline]
Britto, J., Tannahill, D. and Keynes, R. (2002). A critical role for sonic hedgehog signaling in the early expansion of the developing brain. Nat. Neurosci. 5, 103-110.[CrossRef][Medline]
Callejo, A., Torroja, C., Quijada, L. and Guerrero, I.
(2006). Hedgehog lipid modifications are required for Hedgehog
stabilization in the extracellular matrix. Development
133,471
-483.
Casarosa, S., Fode, C. and Guillemot, F. (1999). Mash1 regulates neurogenesis in the ventral telencephalon. Development 126,525 -534.[Abstract]
Caspary, T., Garcia-Garcia, M. J., Huangfu, D., Eggenschwiler, J. T., Wyler, M. R., Rakeman, A. S., Alcorn, H. L. and Anderson, K. V. (2002). Mouse Dispatched homolog1 is required for long-range, but not juxtacrine, Hh signaling. Curr. Biol. 12,1628 -1632.[CrossRef][Medline]
Charron, F., Stein, E., Jeong, J., McMahon, A. P. and Tessier-Lavigne, M. (2003). The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 113,11 -23.[CrossRef][Medline]
Chen, M. H., Li, Y. J., Kawakami, T., Xu, S. M. and Chuang, P.
T. (2004a). Palmitoylation is required for the production of
a soluble multimeric Hedgehog protein complex and long-range signaling in
vertebrates. Genes Dev.
18,641
-659.
Chen, W., Ren, X. R., Nelson, C. D., Barak, L. S., Chen, J. K.,
Beachy, P. A., de Sauvage, F. and Lefkowitz, R. J. (2004b).
Activity-dependent internalization of smoothened mediated by beta-arrestin 2
and GRK2. Science 306,2257
-2260.
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]
Chuang, P. T. and McMahon, A. P. (1999). Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397,617 -621.[CrossRef][Medline]
Chuang, P. T., Kawcak, T. and McMahon, A. P.
(2003). Feedback control of mammalian Hedgehog signaling by the
Hedgehog-binding protein, Hip1, modulates Fgf signaling during branching
morphogenesis of the lung. Genes Dev.
17,342
-347.
Cooper, M. K., Porter, J. A., Young, K. E. and Beachy, P. A.
(1998). Teratogen-mediated inhibition of target tissue response
to Shh signaling. Science
280,1603
-1607.
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.[CrossRef][Medline]
Danesin, C., Agius, E., Escalas, N., Ai, X., Emerson, C.,
Cochard, P. and Soula, C. (2006). Ventral neural progenitors
switch toward an oligodendroglial fate in response to increased Sonic hedgehog
(Shh) activity: involvement of sulfatase 1 in modulating Shh signaling in the
ventral spinal cord. J. Neurosci.
26,5037
-5048.
Dawber, R. J., Hebbes, S., Herpers, B., Docquier, F. and van den Heuvel, M. (2005). Differential range and activity of various forms of the Hedgehog protein. BMC Dev. Biol. 5, 21.[CrossRef][Medline]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[CrossRef][Medline]
Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M. and Edlund, T. (1995). Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube. Cell 81,747 -756.[CrossRef][Medline]
Feng, J., White, B., Tyurina, O. V., Guner, B., Larson, T., Lee,
H. Y., Karlstrom, R. O. and Kohtz, J. D. (2004). Synergistic
and antagonistic roles of the Sonic hedgehog N- and C-terminal lipids.
Development 131,4357
-4370.
Fuccillo, M., Rallu, M., McMahon, A. P. and Fishell, G.
(2004). Temporal requirement for hedgehog signaling in ventral
telencephalic patterning. Development
131,5031
-5040.
Fuccillo, M., Joyner, A. L. and Fishell, G. (2006). Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development. Nat. Rev. Neurosci. 7,772 -783.[CrossRef][Medline]
Furuta, Y., Piston, D. W. and Hogan, B. L. (1997). Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development 124,2203 -2212.[Abstract]
Gallet, A., Ruel, L., Staccini-Lavenant, L. and Therond, P.
P. (2006). Cholesterol modification is necessary for
controlled planar long-range activity of Hedgehog in Drosophila epithelia.
Development 133,407
-418.
Gato, A., Moro, J. A., Alonso, M. I., Bueno, D., De La Mano, A. and Martin, C. (2005). Embryonic cerebrospinal fluid regulates neuroepithelial survival, proliferation, and neurogenesis in chick embryos. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 284,475 -484.[Medline]
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A.
and Scott, M. P. (1996). Conservation of the hedgehog/patched
signaling pathway from flies to mice: induction of a mouse patched gene by
Hedgehog. Genes Dev. 10,301
-312.
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.
Guerrero, I. and Chiang, C. (2006). A conserved mechanism of Hedgehog gradient formation by lipid modifications. Trends Cell Biol. 17,1 -5.[Medline]
Gurdon, J. B. and Bourillot, P. Y. (2001). Morphogen gradient interpretation. Nature 413,797 -803.[CrossRef][Medline]
Hayashi, S., Lewis, P., Pevny, L. and McMahon, A. P. (2002). Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Mech. Dev. 119 Suppl. 1,S97 -S101.[CrossRef][Medline]
Herz, J., Willnow, T. E. and Farese, R. V., Jr (1997). Cholesterol, hedgehog and embryogenesis. Nat. Genet. 15,123 -124.[CrossRef][Medline]
Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Hollway, G. E., Maule, J., Gautier, P., Evans, T. M., Keenan, D. G., Lohs, C., Fischer, D., Wicking, C. and Currie, P. D. (2006). Scube2 mediates Hedgehog signalling in the zebrafish embryo. Dev. Biol. 294,104 -118.[CrossRef][Medline]
Horton, S., Meredith, A., Richardson, J. A. and Johnson, J. E. (1999). Correct coordination of neuronal differentiation events in ventral forebrain requires the bHLH factor MASH1. Mol. Cell. Neurosci. 14,355 -369.[CrossRef][Medline]
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Jullien, J. and Gurdon, J. (2005). Morphogen
gradient interpretation by a regulated trafficking step during ligand-receptor
transduction. Genes Dev.
19,2682
-2694.
Kawakami, T., Kawcak, T., Li, Y. J., Zhang, W., Hu, Y. and Chuang, P. T. (2002). Mouse dispatched mutants fail to distribute hedgehog proteins and are defective in hedgehog signaling. Development 129,5753 -5765.[CrossRef][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.
