Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
* Author for correspondence (e-mail: amcmahon{at}mcb.harvard.edu)
Accepted 8 November 2004
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SUMMARY |
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Key words: Hedgehog signaling, Patched 1 (Ptch1, Ptc1), Hhip (Hip1), Feedback, Mouse
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Introduction |
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One important regulatory strategy used in many morphogen systems is
negative feedback, where signal transduction leads to upregulation of
inhibitors of the pathway to attenuate signaling
(Perrimon and McMahon, 1999;
Freeman, 2000
). Hedgehog (Hh)
family proteins, which are important morphogens in both vertebrate and
invertebrate development, use such feedback regulation where the inhibitor is
actually the Hh receptor, Patched (Ptc), a twelve-pass transmembrane protein
(reviewed by Ingham and McMahon,
2001
). Ptc is a negative regulator of the Hh pathway that exerts
its effects in two different ways: In the absence of Hh, Ptc inhibits activity
of Smoothened (Smo), a downstream activator of the pathway. Binding of Hh to
Ptc abrogates this repression on Smo, allowing Smo-mediated signal
transduction and altered expression of target genes by controlling activities
of the transcriptional effector, Cubitus interruptus (Ci). One of the genes
upregulated by Hh signaling is Ptc itself, and thus Ptc is
expressed at high levels in the Hh target field close to the source of the
ligand (Hooper and Scott,
1989
; Nakano et al.,
1989
). Here, Ptc displays its second function, namely, to
sequester Hh (Chen and Struhl,
1996
). Studies in Drosophila have established that
feedback-mediated upregulation of Ptc is dispensable for
ligand-independent antagonism (LIA), the inhibition of Smo, but is essential
for ligand-dependent antagonism (LDA) that results from Hh sequestration. In
this study, a transgene expressing a low basal level of Ptc from a
heterologous promoter was sufficient to replace endogenous
Ptc-mediated LIA to Smo activity, but failed to restrain the range of
Hh signaling in the responding tissues, indicating that LDA was abolished. LDA
limits the range over which the ligand can diffuse, and thus sharpens the
morphogen gradient (Chen and Struhl,
1996
).
Many features of Hh signaling are conserved between Drosophila and
mouse, including the fact that Ptch1, the mammalian Hh receptor, is a
transcriptional target of the pathway
(Goodrich et al., 1996). The
role of Ptch1-mediated feedback antagonism during mouse development has been
tested in an experiment analogous to the Drosophila experiment
described above; Ptch1-/- embryos, which die around E8.5
with widespread activation of Hh pathway because of loss of LIA, were provided
with a transgene that ubiquitously expresses Ptch1 at low levels from a
metallothionein promoter (MtPtch1)
(Goodrich et al., 1997
;
Milenkovic et al., 1999
). This
transgene was expected to restore LIA but not LDA, as the transgene is not
responsive to Hh signaling. Surprisingly, the resulting embryos
(MtPtch1;Ptch1-/-) exhibited a grossly normal body plan
both externally and internally at E14.5, a result apparently at odds with
Ptch1 playing a significant role in LDA of Hh signaling in the mouse.
Unlike Drosophila, vertebrates have several Hh-binding proteins
that are transcriptionally regulated by Hh signaling; patched 2
(Ptch2) and Hh-interacting protein 1 (Hhip1) are positively
regulated, whereas growth arrest specific gene 1 (Gas1) is negatively
regulated (Carpenter et al.,
1998; Motoyama et al.,
1998
; Chuang and McMahon,
1999
; Lee et al.,
2001
). The role of Ptch2 or Gas1 in Hh-mediated
patterning processes during normal development has yet to be established.
Overexpression and loss-of-function studies in the mouse indicate that Hhip1,
a cell-surface glycoprotein, is an antagonist of Hh signaling;
Hhip1-/- embryos die soon after birth, owing to lung
defects indicative of overactive Hh signaling
(Chuang and McMahon, 1999
;
Chuang et al., 2003
). However,
other parts of the body where Hh signaling plays important roles, e.g. the
limb, face and spinal cord, develop normally in Hhip1 mutants. Taken
together, the mild phenotypes of both MtPtch1;Ptch1-/- and
Hhip1-/- embryos suggest that Ptch1 and Hhip1 may be
functionally redundant in providing feedback LDA to Hh ligands. Consistent
with this view, removing one copy of Ptch1 allele in
Hhip1-/- embryos
(Hhip1-/-;Ptch1+/-) causes earlier lethality
(around E12.5) and more severe lung and pancreas defects than those observed
in Hhip1-/- embryos
(Chuang et al., 2003
;
Kawahira et al., 2003
).
Although the previous studies point to a role for Ptch1 and Hhip1 in
attenuation of paracrine Hh signaling, they did not address the issue of how
LDA might contribute to controlling the magnitude (pathway activity at a given
position in the tissue) or range (total distance over which the pathway is
activated) of a morphogen signaling gradient to generate a specific pattern.
The best evidence for Shh acting as a morphogen comes from studies in the
vertebrate spinal cord (reviewed by
Jessell, 2000;
Briscoe and Ericson, 2001
;
McMahon et al., 2003
). Here,
Shh is first produced from the notochord that underlies the neural tube, and
directs the formation of floor plate which in turn expresses Shh. Shh
from these two ventral midline sources forms a concentration gradient along
the dorsoventral (DV) axis of the neural tube, and represses (Class I
proteins) or induces (Class II proteins) expression of several homeodomain and
basic helix-loop-helix transcription factors at different thresholds.
Cross-repression between the transcription factors sharing a border further
sharpens the boundaries of their territories to define five neural progenitor
domains in the ventral half of the spinal cord (from ventral to dorsal, p3,
pMN, p2, p1, p0; see Fig. 2A).
Finally, cells in each domain differentiate into specific types of neurons
(from ventral to dorsal, V3, motoneuron (MN), V2, V1, V0;
Fig. 2A) based on the
combinations of transcription factors they express. As Hh signaling controls
the specification of individual progenitor domains by a direct and
dose-dependent mechanism (Marti et al.,
1995
; Roelink et al.,
1995
; Ericson et al.,
1997
; Briscoe et al.,
2000
; Briscoe et al.,
2001
; Wijgerde et al.,
2002
), changes in the expression of progenitor domain-associated
transcription factors provide sensitive readouts for any perturbations in the
Shh morphogen gradient.
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Materials and methods |
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Immunofluorescence, in situ hybridization and ß-galactosidase staining on neural tube sections
Embryos were fixed in 4% paraformaldehyde in PBS for 2 hours (E10.5) or 0.5
hours (E8.5), washed in phosphate-buffered saline (PBS) three times for 10
minutes each, cryoprotected overnight in 30% sucrose/0.1 M phosphate buffer
(pH 7.4) and embedded in OCT compound (Tissue-Tek). Frozen sections were
prepared at 10 µm (E10.5) or 12 µm (E8.5). For immunofluorescence
analysis, the sections were blocked in 3% bovine serum albumin, 1%
heat-inactivated sheep serum, 0.1% TritonX-100 in PBS for 1 hour, and then
primary antibodies were applied and sections incubated at 4°C overnight.
Secondary antibodies were applied at room temperature for 1 hour. Where
indicated, the sections were also stained for nuclei (Topro3, Molecular
Probes). The images were collected using a Zeiss LSM510 confocal microscope,
and the sizes of domains or the numbers of cells expressing particular markers
were measured manually from these images. The antibodies used were as follows:
rabbit anti-Olig2 1:5000 (gift of H. Takebayashi), rabbit anti-Nkx6.1 1:3000
(gift of J. Jensen), rabbit anti-En1 1:200 (gift of A. Joyner), rabbit
anti-Foxa2 1:4000 (gift of A. Ruiz I Altaba), mouse anti-Evx1/2 1:100 (gift of
T. Jessell), rabbit anti-Chox10 1:5000 (gift of T. Jessell), rat anti-Lbx1
1:100 (gift of M. Goulding), mouse anti-Nkx2.2 1:50 (DSHB), mouse anti-MNR2
1:20 (DSHB), mouse anti-Pax7 1:20 (DSHB), mouse anti-Pax6 1:20 (DSHB), mouse
anti-Shh 1:25 (DSHB), mouse anti-Lim1/2 1:50 (DSHB), mouse anti-Msx1/2 1:30
(DSHB), and Alexa 488 or 568 goat anti-rabbit, anti-mouse or anti-rat
(Molecular Probes). Section in situ hybridization was performed as described
previously (Schaeren-Wiemers and Gertin-Moser, 1993). The Sim1 probe was a
gift from D. Rowitch. ß-Galactosidase staining was performed using X-gal
as described (Whiting et al.,
1991).
Statistical analysis
Three embryos of each genotype and one section from each embryo (E10.5), or
two embryos of each genotype and two sections from each embryo (E8.5) were
used for quantifications. For the neuronal precursors in
Fig. 4, cells from both sides
of the spinal cord were counted even though only the right halves are shown in
the figure. Difference between wild type and mutant were analyzed by Student's
t-test.
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Transfection and immunostaining of tissue culture cells
Separate dishes of COS-7 cells were transiently transfected with constructs
for Ptch1-YFP, Hhip1-YFP, YFP-GPI or Shh-FLAG using FuGENE6 (Roche) following
manufacturer's instruction. The next day, cells were trypsinized and mixed in
combinations of Ptch1-YFP + Shh-FLAG, Hhip1-YFP + Shh-FLAG or YFP-GPI +
Shh-FLAG, and replated on glass cover slips. After another day of incubation,
cells were permeabilized in 3% paraformaldehyde, 0.5% TritonX-100 for 2
minutes, fixed in 3% paraformaldehyde in PBS for 15 minutes, and blocked in 6%
bovine serum albumin in PBS for 1 hour. Primary antibody (mouse anti-FLAG M2,
Sigma) was applied at a 1:300 dilution for 1 hour, followed by secondary
antibody (Alexa 568 goat anti-mouse, Molecular Probes) at 1:400 for 30
minutes. Nuclei were stained with Topro3 (Molecular Probes), and the images
were collected using a Zeiss LSM510 confocal microscope.
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Results |
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The Hhip1-/- neural tube was similar to the wild type in overall size (data not shown), whereas Hhip1-/-;Ptch1+/- neural tube was enlarged, consistent with the increased body size [Fig. 2B,C; neural tube cross-section area: wild type, 0.137 (±0.013) mm2; mutant, 0.184 (±0.019) mm2; P=0.062]. Patterning of the ventricular zone was grossly normal in both the Hhip1-/- and Hhip1-/-;Ptch1+/- neural tube (Fig. 2D-R); in the latter, both ventral (Fig. 2D-O,S) and dorsal (Fig. 2P-R) cell types were expanded, thus preserving the relative proportions between dorsal and ventral domains to some degree [size of dorsal domain along DV axis: wild type, 45(±4.04) cell diameters; Hhip1-/-;Ptch1+/-, 56(±7.37) cell diameters; P=0.053]. However, quantitative analysis revealed a small but statistically significant expansion of ventral progenitor domains in both genotypes (Fig. 2T,U). For example, when the differences in neural tube size are taken into account (Fig. 2U), the domain expressing Nkx6.1 (Nkx6.1+, p3+pMN+p2), which normally occupies 20(±1.08)% of the ventral neural tube, is enlarged to 25.3(±2.25)% (P=0.031) and 29.3(±3.84)% (P=0.033) in Hhip1-/- and Hhip1-/-;Ptch1+/- embryos, respectively.
A complete loss of Ptch1- and Hhip1-mediated LDA leads to a dramatic expansion of ventral progenitor domains, and a reduction of intermediate and dorsal progenitor domains
Next, we examined effects of eliminating Ptch1 component of LDA using
MtPtch1;Ptch1-/- embryos. In their neural tube, we
observed a 2.9-fold expansion of Nkx2.2+ p3 domain
[Fig. 3I,J,Z; size of p3 along
D-V axis: wild type, 5(±1) cell diameters; mutant, 14(±3.5) cell
diameters; P=0.057] and 1.4-fold expansion of the Olig2+
pMN domain [Fig. 3I,J,Z; size
of pMN along DV axis: wild type, 4(±1.2) cell diameters; mutant,
6(±1) cell diameters; P=0.038], although it had less of an
effect in more dorsal region (Fig.
3M,N,Q,R,Z,a). Thus, expression of Ptch1 from MtPtch1 is
not able to fully compensate for the removal of endogenous Ptch1, and an
enhanced response is observed in the ventral-most Shh-dependent cell
identities. However, removing one copy of Hhip1
(MtPtch1;Ptch1-/-;Hhip1+/-) resulted in a
further increase in the size of the p3 domain
[Fig. 3I,K,Z; size of p3 along
DV axis: wild type, 5(±1) cell diameters; mutant, 17(±1.5) cell
diameters; P=0.003]. The most striking patterning defects were
observed in MtPtch1;Ptch1-/-;Hhip1-/- embryos.
In these mutants, the floor plate was three times larger than that of
wild-type embryos (Fig.
3A,D,E,H,Y; number of Foxa2+ cells: wild type,
23±1; mutant, 69±39; P=0.172). Furthermore, p3 and pMN
domains expanded by 5.3 and 3.2 fold, respectively
[Fig. 3I,L,Z; size of p3 along
DV axis: wild type, 5(±1) cell diameters; mutant, 27(±5.9) cell
diameters; P=0.017; size of pMN along DV axis: wild type,
4(±1.2) cell diameters; mutant, 14(±1.7) cell diameters;
P=0.028]. By contrast, the Nkx6.1-/Pax7- p1+p0
domain was reduced by 3.7 fold [Fig.
3M,P,Z; size of p1+p0 along DV axis: wild type, 14(±2.1)
cell diameters; mutant, 4(±3.5) cell diameters; P=0.010]. The
size of Pax7+ dorsal domain was also dramatically decreased, by 3.6
fold (Fig. 3Q,T,Z; size of
dorsal domain along DV axis: wild type, 45(±4.0) cell diameters;
mutant, 13(±8.7) cell diameters; P=0.007). The dorsalmost
cells in the neural tube normally express Msx1/2 in response to BMP signaling
from the roof plate and surface ectoderm
(Liem et al., 1995).
Immunostaining for Msx1/2 proteins indicated that this dorsal signaling is
maintained in even the most extreme genotype,
MtPtch1;Ptch1-/-;Hhip1-/-
(Fig. 3U-X).
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Reduction in intermediate and dorsal neuronal precursors in the neural tube of MtPtch1;Ptch1-/-;Hhip1-/- embryos
We examined whether the changes in neural progenitor domains caused by
compromised LDA is reflected by the populations of post-mitotic neuronal
precursors. Seven markers were used to identify different neuronal precursor
subtypes; in a ventral-to-dorsal progression, these were Sim1 (V3), MNR2 (MN),
Chox10 (V2), En1 (V1), Evx1/2 (V0), Lbx1 (dI4+5+6) and Lim1/2 (dI2, dorsal to
Lbx1) (Fig. 2A;
Fig. 4A,G,M,S).
In Hhip1-/- embryos, all seven groups of neuronal precursors were present at normal numbers except for a moderate decrease (37.5%) in V2 cells (Fig. 4A,B,G,H,M,N,S,T,Y-e). However, Hhip1-/-;Ptch1+/- embryos showed a general expansion of neuronal precursor populations (except for dI2), consistent with their proportionally enlarged neural tube reported earlier (Fig. 4C,I,O,U,Y-e).
In MtPtch1;Ptch1-/- embryos, the V3 population was twice that of wild-type embryos (Fig. 4A,D,Y; Sim1+ area on the section: wild type, 24.7(±3.76) (x100 µm2); mutant, 48(±5) (x100 µm2); P=0.003). By contrast, MN, V2, V1, V0 and dI4+5+6 populations did not change significantly (Fig. 4G,J,M,P,S,V,Z-d), but dI2 precursors were reduced by 40% in the absence of Ptch1 LDA [Fig. 4S,V,e; dI2: wild type, 50(±5.3) cells; mutant, 30(±3.5) cells; P=0.003]. A more severe alteration in neuronal precursor populations was observed when Hhip1 activity was attenuated or removed on this background. In MtPtch1;Ptch1-/-;Hhip1+/- embryos, V3 precursors expanded by 2.2-fold compared with the wild type [Fig. 4A,E,Y; Sim1+ area on the section: wild type, 24.7(±3.76) (x100 µm2); mutant, 55(±4) (x100 µm2); P=0.001]. The MN population also underwent a 1.5-fold increase [Fig. 4G,K,Z; MN: wild type, 159(±30.4) cells; mutant, 244(±22.1) cells; P=0.031]. By contrast, while V2, V1, V0, and dI4+5+6 precursors were all reduced, these changes were not statistically significant (Fig. 4G,K,M,Q,S,W,a-d). A more severe decrease in dI2 precursors than that observed in MtPtch1;Ptch1-/- embryos (2.3-fold fewer than normal) was apparent in the MtPtch1;Ptch1-/-;Hhip1+/- neural tube [Fig. 4S,W,e; dI2: wild type, 50(±5.3) cells; mutant, 21(±7.4) cells; P=0.004].
MtPtch1;Ptch1-/-;Hhip1-/- embryos showed the most striking changes in neuronal precursor populations, as expected from their severe defects in progenitor domain patterning. V3 precursors in these mutants were increased by 2.3 fold compared with wild-type embryos [Fig. 4A,F,Y; V3: wild type, 24.7(±3.76) (x100 µm2); mutant, 57(±2) (x100 µm2); P=0.010]. The MN population was not significantly different from the wild type (Fig. 4G,L,Z). However, V2 precursors were reduced by 5.3 fold [Fig. 4G,L,a; V2: wild type, 16(±0) cells; mutant, 3(±2) cells; P=0.008], and intermediate (V1, V0) and dorsal (dI4+5+6, dI2) neuronal precursors were all present at less than 10% of their normal numbers [Fig. 4M,R,S,X,b-e; V1: wild type, 42(±9.0) cells; mutant, 3(±4.2) cells; P=0.016; V0: wild type, 32(±11.7) cells; mutant, 3(±3.8) cells; P=0.076; dI4+5+6: wild type, 72(±19.3) cells; mutant, 5(±6.11) cells; P=0.035; dI2, wild type, 50(±5.29) cells; mutant, 4(±3.8) cells; P=0.003]. Figure 4f shows the relative sizes of neuronal precursor populations among wild type, MtPtch1;Ptch1-/-, MtPtch1;Ptch1-/-;Hhip1+/- and MtPtch1;Ptch1-/-;Hhip1-/- embryos for the seven groups of neuronal precursors analyzed. Clearly, in the absence of feedback LDA by Ptch1 and Hhip1 (MtPtch1;Ptch1-/-;Hhip1-/-), the number of the ventral neuronal precursors (V3) increased, while intermediate (V1, V0) and dorsal (dI4+5+6, dI2) neuronal precursors were almost ablated, a result consistent with the expansion of ventral progenitor domains and corresponding reduction of intermediate and dorsal progenitor domains in this genotype (Fig. 3Z,a).
The basic patterning of the ventral neural tube is specified at early stages by notochord-derived Shh
We originally predicted that even though ligand production is unaltered,
removing LDA would result in both enhanced Hh signaling as increased amount of
ligand should be available to trigger receptor-mediated signaling, and an
increased spatial range of ligand action in the target field. However, one of
the phenotypes we observed in
MtPtch1;Ptch1-/-;Hhip1-/- embryos was a
threefold increase in size of the floor plate, which itself expresses
Shh. This raised the possibility that the patterning defects we
observed were due to an increase in ligand production, rather than diffusion
or availability of Shh.
During embryogenesis, Shh is first expressed in the notochord and
then signals to the overlying neural tube to induce its own expression in the
floor plate. Although it has been recognized that the acquisition of general
ventral identity by neural tube cells depends on early Hh signaling from
notochord before the floor plate is established
(Ericson et al., 1996), the
detailed kinetics for each of Shh-mediated patterning events has not been
rigorously addressed to date. We examined early stages of neural tube
development (E8.5), at the axial level of the second to fourth somite from the
anterior end, to determine timing of ventral progenitor domain patterning in
relation to induction of Shh in the floor plate
(Fig. 5). In a mouse embryo,
one somite is added approximately every 90-120 minutes, and thus somitogenesis
provides a convenient temporal staging mechanism. Expression of
Nkx6.1 and repression of a Class I gene Pax6 was already
evident at the four-somite stage, the earliest time point we examined
(Fig. 5M-R). Cells with weak
Olig2 production were first identified at the six-somite stage, but the Olig2
level and the number of Olig2-producing cells increased significantly by the
eight-somite stage (Fig. 5G-L).
Nkx2.2 was present from the eight-somite stage
(Fig. 5G-L). Although
Foxa2-producing cells were also detected in the eight-somite stage embryo,
high-level expression was not evident until the 13-somite stage
(Fig. 5A-F). Importantly, while
Shh was expressed in the notochord at all stages examined (arrows in
Fig. 5A-F), we observed Shh
production by the floor plate only at the 16-somite stage
(Fig. 5A-F), shortly after
high-level Foxa2 expression was established in ventral midline cells
(Fig. 5E). These results
indicate that the basic organization of ventral neural progenitor domains is
set up by the eight- to 10-somite stage as a result of Shh signaling from the
notochord, well before Shh expression commences in the floor plate.
This finding is consistent with previous reports that Gli2 mutants,
which lack a floor plate, preserve all other ventral cell types, although the
p3 progenitor number is decreased (Ding et
al., 1998
; Matise et al.,
1998
). Furthermore, the results in
Fig. 5 suggest that there is a
temporal sequence of repression of Class I and activation of Class II genes at
the ventral midline in response to Shh signaling that mirrors their
concentration thresholds observed in vitro
(Ericson et al., 1997
;
Briscoe et al., 2000
).
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In the Ptch1-null allele, E. coli lacZ gene encoding
ß-galactosidase (ß-gal) was placed under the endogenous
Ptch1 promoter, providing a reporter for Hh pathway activation
(Ptch1LacZ) (Goodrich
et al., 1997). In the Ptch1+/- neural tube,
which is phenotypically wild type, ß-gal activity was present in cells at
the ventral end and tapered off in dorsal cells, probably reflecting a normal
Shh morphogen gradient (Fig.
6S) (Goodrich et al.,
1997
). In the Ptch1-/- neural tube, a uniform
high-level of ß-gal activity along the entire DV axis indicated that
ubiquitous, ligand-independent activation of Hh pathway was occurring
(Fig. 6X) (Goodrich et al., 1997
).
However, the introduction of an MtPtch1 transgene onto this
background (MtPtch1;Ptch1-/-) eliminated ectopic
Ptch1lacZ expression in the dorsal half of the neural tube
and restored the DV gradient of staining although the stained domain was
expanded (Fig. 6V). Thus,
MtPtch1-mediated LIA was clearly sufficient to block Hh pathway
activation where there was no ligand, and the enhanced Hh signaling observed
in the ventral half of the neural tube reflects a compromise in LDA. When both
Ptch1- and Hhip1-mediated LDA were removed
(MtPtch1;Ptch1-/-;Hhip1-/-), ß-gal
expression extended along the entire DV axis of the neural tube
(Fig. 6W), but unlike the
expression in Ptch1-/- embryos, where ß-gal activity
was uniform, a ventral (high) to dorsal (low) gradient of ß-gal activity
was retained in the MtPtch1;Ptch1-/-;Hhip1-/-
neural tube. As it is very likely that all Hh signaling in the
MtPtch1;Ptch1-/-;Hhip1-/- neural tube is ligand
dependent (see Discussion), the extent of ß-gal and Nkx6.1 expression
(Fig. 6Q,W) provides compelling
evidence that Shh can travel from the notochord along the entire DV extent of
the neuraxis if its movement is not impeded by LDA.
Distinct subcellular localizations of Shh-Ptch1 and Shh-Hhip1 complexes suggest different molecular mechanisms for Ptch1- and Hhip1-mediated LDA
Given their functional overlap in LDA, we examined Ptch1 and Hhip1
activities in cell culture to determine whether both factors use the same
mechanism to perform this function. To address this issue, we transfected two
populations of COS-7 cells independently with constructs for Ptch1 tagged with
yellow fluorescent protein (Ptch1-YFP) and Shh tagged with FLAG epitope
(Shh-FLAG), and then mixed the transfected cells on the same dish. We focused
our analysis on situations where cells expressing Ptch1-YFP were next to those
expressing Shh-FLAG. In these cases, the interaction between the two proteins
was not detected at the cell surface (see Fig. S1A,G,M in the supplementary
material), but co-localization was observed in intracellular vesicles within
Ptch1-YFP producing cells (arrowheads in Fig. S1B,H,N in the supplementary
material). These results are consistent with a previous report that
Ptch1-mediated LDA appears to remove Shh ligand from the extracellular space
by rapid endocytosis, which is probably followed by lysosomal degradation of
the ligand (Incardona et al.,
2000). In contrast to Ptch1, we observed a strong co-localization
of Hhip1-YFP and Shh-FLAG at the cell surface in a similar experiment (see
Fig. S1C,I,O in the supplementary material). In particular, we found a strong
accumulation of Hhip1-YFP along the edge of the cells abutting those
expressing Shh-FLAG (see Fig. S1 in the supplementary material). A small
amount of Shh-FLAG was also found in intracellular vesicles in Hhip1-YFP
expressing cells, where the two proteins co-localized (arrowheads in Fig.
S1D,J,P in the supplementary material), but this was a very minor fraction
compared with the level of proteins on the cell surface. A control
heterologous cell surface protein (YFP with glycosylphosphatidylinositol (GPI)
anchor) did not colocalize with Shh either on the cell surface (see Fig.
S1E,K,Q in the supplementary material) or intracellularly (see Fig. S1F,L,R in
the supplementary material). Thus, Hhip1 may prevent interaction of Shh with
its receptor by sequestering ligand at the cell surface. Endocytosis of Hhip1
and Shh complex, albeit slow, may ultimately remove some ligand-Hhip1
complexes.
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Discussion |
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That a feedback control influences movement of Shh in the neural tube has
been postulated from other approaches. When clones of cells that were unable
to transduce Hh signal were introduced in the ventral neural tube, cells just
dorsal to these clones showed signs that they were exposed to higher levels of
Shh than they normally receive (Briscoe et
al., 2001; Wijgerde et al.,
2002
). However, these studies could not specifically address the
role of Ptch1 and Hhip1 in LDA, as these clones are unable to express any of
the genes that are induced by Hh signaling, among which may be other
unidentified antagonists to Shh action.
With regards to the biological significance of LDA in the context of development, removing both Ptch1- and Hhip1-mediated feedback LDA caused a dramatic expansion of ventral progenitors (p3, pMN) at the expense of intermediate (p0+p1) or dorsal progenitors in the neural tube (Fig. 3Z,a). This patterning defect resulted in the generation of an abnormally large number of ventral neurons (V3) and an almost complete lack of intermediate (V1, V0) or dorsal (dI4+5+6, dI2) neurons (Fig. 4f). This result demonstrates that feedback control on Shh signaling by LDA is crucial to the formation of appropriate numbers of all types of ventral and intermediate neurons in the developing spinal cord. Furthermore, the early lethality (around E10.5) and gross abnormalities in the brain and face of MtPtch1;Ptch1-/-;Hhip1-/- embryos suggest that this regulatory mechanism plays important roles in many other regions of the embryo as well.
A recent study using mathematical modeling proposed that the self-enhanced
ligand degradation, such as the one resulting from feedback LDA of Ptc (and
perhaps Hhip1) to Hh, is essential for robustness of the morphogen gradients
to fluctuations in ligand production
(Eldar et al., 2003). This
prediction can be tested by changing doses of Shh allele in embryos
with normal or compromised feedback LDA and comparing the changes in the
neural tube patterning. For example, differences between the
MtPtch1;Ptch1-/-;Hhip1-/- and
MtPtch1;Ptch1-/-;Hhip1-/-;Shh+/-
neural tubes are expected to be larger than those between the wild-type and
Shh+/- neural tubes.
Neural tube patterning defects in the absence of feedback LDA to Shh
In the MtPtch1;Ptch1-/-;Hhip1-/- neural
tube, the cell types induced by high levels of Hh signaling, i.e. floor plate,
p3 and pMN, all expanded by three- to fivefold, consistent with an overactive
Hh pathway in these embryos. However, in the same mutants, p2 progenitor
numbers did not change significantly, and p1+p0 progenitors were markedly
reduced by almost fourfold. Given that p2 and p1+p0 populations are also
directly dependent on and induced by Hh signaling, albeit at lower
concentration thresholds than either floor plate, p3 or pMN progenitors, why
did they fail to expand (Pierani et al.,
1999; Briscoe et al.,
2001
; Wijgerde et al.,
2002
)? The answer probably lies in the effects of BMP signals on
specification of ventral cell types. BMPs can desensitize ventral neural cells
to Shh by interfering with intracellular signal transduction of the Hh pathway
(Liem et al., 2000
).
Furthermore, Dbx1 and Dbx2, two transcription factors that demarcate p1 and p0
domains, are repressed by high levels of BMP signaling
(Pierani et al., 1999
). In the
normal spinal cord, BMPs are expressed at the surface ectoderm and roof plate
at the dorsal midline (Liem et al.,
1995
; Liem et al.,
1997
), while BMP inhibitors noggin and follistatin are expressed
ventrally to counter the actions of BMPs
(McMahon et al., 1998
;
Liem et al., 2000
). In
MtPtch1;Ptch1-/-;Hhip1-/- embryos, as Shh
signaling is enhanced, the low levels of pathway activation appropriate for p2
to p0 fates are likely to occur in a relatively more dorsal position than
normal, bringing them close to the dorsal midline
(Fig. 3Z). Analysis of Msx1/2
expression indicates that BMP signaling here is intact in
MtPtch1;Ptch1-/-;Hhip1-/- embryos
(Fig. 3X). Therefore,
prospective p2-p0 populations are constrained by too high Hh signaling
ventrally and the inhibitory action of BMP signaling dorsally.
The Pax7+ dorsal progenitor domain was severely reduced in the MtPtch1;Ptch1-/-;Hhip1-/- neural tube. This result supports the idea that the expansion of ventral cell types we observed was at the expense of dorsal cell fates (i.e. cells that would normally adopt a dorsal fate instead adopted a ventral fate), and argues against an alternative explanation that appropriate numbers of ventral and dorsal neural progenitors were specified initially, but enhanced Hh signaling increased the proliferation rates of ventral cells, causing abnormally rapid expansion of the ventral domains while the dorsal domains grow at the normal rate. In the latter scenario, the size of the dorsal domain in the mutant would remain similar to that of the wild type. Compelling support for the model of direct specification defects comes from the fact that the severe patterning phenotypes of MtPtch1;Ptch1-/-;Hhip1-/- neural tube were obvious at E8.5 within a few hours of the onset of Shh signaling, insufficient time for a several-fold expansion of a progenitor population by cell proliferation. Furthermore, if the changes in cell proliferation or survival rates were the cause, then the patterning phenotype is expected to get worse as cells go through additional division cycles over the following 2 days. Rather, the ventralization of the mutant neural tube at E8.5 was as severe as it was at E10.5 (compare Fig. 3Z,a and Fig. 6Y,Z).
Mechanism of Ptch1- and Hhip1-mediated LDA to Shh
Although our genetic analysis established that Ptch1 and Hhip1 play
overlapping roles in providing LDA to Shh (and most probably other Hh signals
in other contexts), the two proteins have very different structures; Ptch1 is
a twelve-pass transmembrane protein with two large extracellular loops, a
C-terminal cytoplasmic domain and a sterol-sensing domain (SSD), a feature
found in several proteins involved in cholesterol homeostasis
(Stone et al., 1996;
Carstea et al., 1997
). By
contrast, Hhip1 has one large extracellular domain anchored to the cell
membrane by a hydrophobic stretch at the C terminus
(Chuang and McMahon, 1999
).
These differences prompted us to compare the molecular mechanisms that Ptch1
and Hhip1 use to inhibit Shh.
Results herein and those of Incardona et al.
(Incardona et al., 2000)
suggest that Ptch1 induces rapid endocytosis of Shh, which is followed by
degradation of the ligand within the lysosome. As exposing cells that are
unable to upregulate Ptch1 expression in response to Hh signal to Hh leads to
a significant net decrease in Ptch1 levels on the cell surface, recycling of
Ptch1 back to the cell surface, if it occurs, does not seem to be very
efficient (Denef et al.,
2000
). Contrary to Ptch1, our results indicate that Hhip1 appears
to inhibit Shh mainly by physically sequestering it at the cell surface, as
the ligand is only very inefficiently internalized. This mechanism implies
that the antagonism of Hhip1 to Shh is stoichiometric, in line with the copy
number-dependent antagonism observed in MtPtch1;Ptch1-/-,
MtPtch1;Ptch1-/-;Hhip1+/- and
MtPtch1;Ptch1-/-;Hhip1-/- embryos. However, the
endogenous expression level of Hhip1 appears to be very low, as
judged from RNA in situ hybridization or Hhip1LacZ
reporter staining (Fig. 6T)
(Chuang and McMahon 1999
),
which is unusual for a factor that acts in a stoichiometric fashion. Direct
quantitative analysis of Shh and Hhip1 protein levels in the target field, a
challenging goal, will be necessary to finally understand this issue.
Growth control by feedback LDA to Hh signaling
Our data that Hhip1-/-;Ptch1+/- embryos had
a larger neural tube than wild type implicate feedback LDA in regulation of
spinal cord growth. The size of an organ is regulated by controlling both cell
death and cell proliferation. In the early neural tube, Shh and Ptch1 have
been shown to have anti-apoptotic and pro-apoptotic effects, respectively
(Charrier et al., 2001;
Thibert et al., 2003
). Later
in development, Shh acts as a mitogen in several areas of the central nervous
system (Rowitch et al., 1999
;
McMahon et al., 2003
).
However, we were unable to detect any significant changes in mitosis or
apoptosis in Hhip1-/-;Ptch1+/- neural tube
compared with the wild type at E10.5 (data not shown). Therefore, it appears
that the growth phenotype of these embryos was due to small increase in
proliferation or survival rates that had cumulative effects over time.
Importantly, for many of Hhip1-/-;Ptch1+/-
embryos, the entire body was overgrown, and this phenotype included an
expansion of both ventral Shh-dependent and dorsal Shh-independent cell
identities in the neural tube. Although Ptch1+/- adult
mice were reported to be larger (Goodrich
et al., 1997), this was not evident at E10.5 (data not shown).
Thus, the growth phenotype of Hhip1-/-;Ptch1+/-
clearly implicates Hhip1 as well as Ptch1 in a systemic process that controls
body size. This is an intriguing result considering that only a small part of
the embryo at E10.5 is subject to Hh signaling and Hhip1 expression
is entirely restricted to cells responding to Hh signals
(Chuang and McMahon, 1999
). As
Hh signaling does not seem to occur in dorsal progenitor domain in
Hhip1-/-;Ptch1+/- embryos based on the
Ptch1LacZ reporter assay
(Fig. 6U), enhanced growth in
this domain most likely involves some other factors. Further study is
necessary to elucidate possible connections between Hh signaling and systemic
growth control mechanism in embryonic and adult mouse. In addition, while our
data provide unambiguous evidence for combined roles of Ptch1 and
Hhip1 in LDA during spinal cord patterning, potential interaction of
these components with Ptch2 and Gas1 remains an unanswered
question that will require additional genetic analysis in future studies.
Furthermore, new approaches may enable these mechanisms to be addressed in
other regions where graded Shh signaling may play a central role in
patterning, most notably digit patterning in the vertebrate limb.
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ACKNOWLEDGMENTS |
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