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First published online 13 February 2008
doi: 10.1242/dev.013086
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Department of Molecular and Cellular Biology, The Biolabs, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
Author for correspondence (e-mail:
mcmahon{at}mcb.harvard.edu)
Accepted 15 December 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Shh, Morphogen, Neural tube pattern, Basal body, Primary cilium
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
developing spinal cord, or neural tube, begins as a folded sheet of neural
progenitor cells that is induced and patterned into spatially distinct domains
by adjacent dorsal and ventral organizing centers: the non-neural ectoderm and
notochord, respectively. Sonic hedgehog (Shh), a member of the Hedgehog (Hh)
family of secreted signaling proteins, mediates the long-range patterning
activity of the notochord and specifies five discrete domains of neural
progenitors and a second organizing center of Shh activity within the
non-neuronal floor plate (FP) at the ventral midline of the neural tube
(Briscoe and Ericson, 2001;
Jessell, 2000;
McMahon et al., 2003).
Increasing concentrations of Shh specify progressively more-ventral cell
types in chick neural explants (Briscoe et
al., 2000). Importantly, FP-deficient Gli2 mutants only
produce Shh from the notochord, but are still capable of specifying all
primary Shh-dependent neural progenitor populations, although some are reduced
in number (Matise et al.,
1998). These findings support a model wherein a graded Shh
signaling response to notochord-derived Shh activates a long-range patterning
process in the ventral neural tube, resulting in the regional organization of
specific neural progenitor types in spatially distinct ventral domains.
However, our understanding of the mechanisms that regulate Shh signal
distribution and response throughout the patterning process is largely
speculative.
To directly visualize Shh during neural tube patterning, we engineered the mouse Shh locus to encode a Shh::GFP fusion protein and examined its distribution in conjunction with Shh-mediated patterning of the ventral neural tube. We report that Shh ligand from the notochord forms a dynamic gradient in the neural target field coincident with the emergence of the ventral pattern, and concentrates adjacent to the apically localized basal body of neural progenitors during this process. Further, we provide evidence that Shh might traffic into the neural target field through ventral midline cells via a microtubule scaffold that spans between the Shh-producing notochord and apically located basal body. These results provide fresh insight into the possible mechanisms of Shh signal distribution and transduction during neural tube patterning.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3x
optical oversampling). The apical region is defined as the space between the
most apically positioned nuclei of the ventral neural tube.
Tissue section preparation and staining
Embryos were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) in
Dulbecco's phosphate-buffered saline (DPBS; Gibco BRL) for 15 minutes (E9.5
and E10.5) or 10 minutes (E8.5), washed in DPBS containing 0.1 M glycine
(DPBS-G) three times for 5 minutes each, cryoprotected 4 hours to overnight in
30% sucrose in DPBS at 4°C, embedded in OCT compound (Tissue-Tek), frozen
on dry ice and stored at -80°C. Frozen sections were cut at 20 µm,
immediately placed in cold DPBS, fixed in cold 4% paraformaldehyde in DPBS for
10 minutes, washed in cold DPBS-G three times for 5 minutes each and stored at
4°C. E10.5 embryos were sectioned at the level of the forelimb and E8.5
embryos at the level of the heart. All sections are displayed such that dorsal
is at the top and ventral is at the bottom. Antibody staining for homeodomain
proteins was performed as described (Jeong
and McMahon, 2005). For other antibodies, sections were incubated
with primary antibodies in block buffer (3% bovine serum albumin, 1%
heat-inactivated sheep serum in DPBS) for 2 hours (room temperature) to
overnight (4°C), washed three times in cold DPBS, incubated with secondary
antibodies in block buffer for 1 hour (room temperature), washed three times
in cold DPBS and mounted in Vectashield hard-set mounting media (Vector Labs)
overnight at room temperature. Antibodies and dilutions were as follows: mouse
anti-Nkx2.2, -Pax6, -Pax7 1:20 (DSHB), rabbit anti-Olig2 1:5000 (gift of H.
Takebayashi, National Institute for Physiological Sciences, Okazaki, Japan),
rabbit anti-Nkx6.1 1:3000 (gift of J. Jensen, Hagedorn Research Institute,
Gentofte, Denmark), rabbit anti-polaris 1:500 (gift of B. Yoder, University of
Alabama, Birmingham, AL), mouse anti-
-tubulin 1:500 (Sigma), mouse
anti-acetylated tubulin 1:2500 (Sigma) and secondary antibodies (Molecular
Probes). Nuclei were stained with Hoechst. In situ hybridization was performed
according to routine procedures.
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) was used
to activate expression of a floxed Rosa-Wnt1::gfp in the
Shh-expressing domain (Carroll et
al., 2005). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Shh acts as a morphogen in ventral patterning of the vertebrate neural tube
(Ingham and Placzek, 2006). To
assess Shh::GFP patterning activity, we assayed Shh::gfp homozygous
neural tubes at E10.5 for Shh-dependent neural progenitor domains. All domains
were present in their correct relative position, although some were reduced in
size (Fig. 1B-D,H). In
addition, Shh::gfp was expressed at the ventral midline
(Fig. 1E-G), indicating that
Shh::GFP induces FP, the cell type known to require the highest level of Shh
activity for its specification (Ericson et
al., 1997; Marti et al.,
1995; Roelink et al.,
1995). Importantly, removing one copy of Ptch1, a
feedback antagonist of Hh signaling that sequesters ligand, significantly
normalized the size of each neural progenitor domain
(Fig. 1H) and the gross
phenotype of Shh::gfp homozygous embryos (see Fig. S3 in the
supplementary material), consistent with the view that GFP modification
results in reduced levels of Shh ligand with normal bioactivity.
|
).
Together, our data indicate that Shh::GFP supports Shh morphogen-based
patterning of the ventral neural tube and has a similar distribution to the
wild-type signaling protein in the context of ventral neural tube
patterning. In order to correlate the distribution of Shh::GFP ligand with Shh-dependent patterning, we focused all subsequent analyses on Shh::gfp homozygous embryos, though generally similar results were observed in more-limited studies of heterozygous embryos (data not shown).
Shh::GFP ligand from the notochord establishes a gradient at the apical region of ventral progenitors
All Shh-dependent progenitor domains are established and the notochord has
regressed by E10.5. Therefore, we examined the Shh::GFP ligand distribution at
earlier stages when active ventral neural patterning is occurring. At E8.5 and
E9.5, the notochord is in direct contact with the basal surface of the neural
tube at the ventral midline and Shh-dependent patterning has initiated but is
not complete (Fig. 2G,H). As
expected, Shh::gfp expression was restricted to the notochord at E8.5
(Fig. 2A), but was observed in
the notochord and at the ventral midline of the neural tube at E9.5
(Fig. 2B). Whereas Shh::GFP
ligand was detected both apically (towards the lumen) and basolaterally at
E9.5 (Fig. 2E), Shh::GFP ligand
was only detected apically at E8.5, when Shh derives exclusively from the
notochord (Fig. 2D). As GFP
detection may be more sensitive than RNA detection, we examined the Shh::GFP
distribution in Gli2 mutant neural tubes, which lack the
Shh-expressing FP population (Matise et
al., 1998), to determine whether the apically localized ligand at
E8.5 might reflect low-level expression of Shh::gfp within ventral
midline neural progenitors. As expected, Shh::gfp expression was
confined to the notochord of Gli2 mutant neural tubes
(Fig. 2C), whereas active
patterning was observed over several cell diameters
(Fig. 2I). Strikingly, Shh::GFP
ligand was only detected in the apical compartment of target cells
(Fig. 2F), as in E8.5 embryos
that have wild-type Gli2 function (Fig.
2D). Thus, Shh::GFP ligand from the notochord concentrates within
an apical subcompartment of neural progenitors coincident with the
specification of ventral cell identities. Importantly, immunodetection of
wild-type Shh protein with an antibody indicated that the distribution of
Shh::GFP ligand reflects the normal distribution of secreted, wild-type Shh
protein (Fig. 2J).
To determine whether the target field accumulation of ligand was a general
property of a protein secreted by the notochord or a more specific property of
Shh, we activated Wnt1::gfp expression in the notochord and examined
the Wnt1::GFP ligand distribution at E8.5 in the overlying neural tube (see
Materials and methods). Like Hh proteins, Wnt proteins are secreted
lipid-modified signaling ligands (Takada
et al., 2006; Willert et al.,
2003). Wnt1::GFP is biologically active
(Carroll et al., 2005) and
Wnt1, along with other Wnt signals, can activate a potent mitogenic response
in neural progenitor cells (Dickinson et
al., 1994; Megason and
McMahon, 2002). In contrast to Shh::GFP, notochord-derived
Wnt1::GFP ligand showed no significant apical accumulation. Instead, Wnt1::GFP
ligand was broadly distributed on or around ventral progenitor cells
(Fig. 2K,L). Thus, the observed
apical localization of Shh within its target field is a Shh-specific
distribution.
|
). The amount of Shh::GFP ligand was found to be
increased in the ventral neural tube, but continued to be predominantly apical
in neural progenitors (Fig.
3D). High magnification of the apical region revealed that small,
individual puncta could still be resolved at a dorsal position
(Fig. 3G, white arrow), as in
8- to 12-somite-stage neural tubes (Fig.
2D). However, Shh::GFP ligand at a more ventral position formed
one large punctate mass, presumably as a result of several overlapping smaller
puncta in this region (Fig. 3G,
red arrow).
Next, we plotted the Shh::GFP ligand distribution within the apical region
along the dorsal-ventral (D-V) axis beginning at the apical surface of ventral
midline cells (Fig. 3J).
Shh::GFP ligand spanned 25 µm, crossing five cell diameters (5 µm
per cell). Mapping the position of Shh-dependent ventral progenitor domains
present at this time (p3, pMN and p2), we found that the concentrated ligand
is highest at p3, drops rapidly across pMN and then slowly disappears across
p2. Thus, apically concentrated Shh::GFP ligand extends as a gradient that
drops exponentially along the D-V axis, spanning three Shh-dependent cell
types.
Shh morphogen activity in the neural tube is regulated by a partially
redundant, Shh-induced negative-feedback mechanism that acts to bind and
sequester Shh ligand at the cell surface
(Jeong and McMahon, 2005). To
determine whether the target field gradient is also regulated by
negative-feedback mechanisms, we quantified the Shh::GFP ligand distribution
in smoothened (Smo) mutant neural tubes, where all Hh-responsiveness
is lost (Zhang et al., 2001).
As expected, Smo neural tubes lacked Shh-dependent ventral
progenitors (Fig. 3A,B).
However, Shh::GFP ligand continued to concentrate within the apical region of
target cells (Fig. 3D,E),
suggesting that this distribution is feedback-independent. However, the
Shh::GFP ligand distribution in Smo mutant neural tubes became
generally less restricted. Significant ligand accumulation was observed on, or
close to, the cell surface of neural progenitors, reminiscent of the
distribution observed in non-responsive neurons within the mantle region in
the E10.5 neural tube (arrow in Fig.
1J) and that observed for notochord-derived Wnt1::GFP ligand
(Fig. 2K).
Quantitative analysis of the apical target field distribution indicated
that the gradient spans over 60 µm (or twelve cell diameters) in the
absence of Smo function, approximately twice as far dorsally as
ligand in neural tubes where Smo-dependent feedback processes are operative
(Fig. 3G,H,J). Further, the
spatial profile of ligand distribution along the D-V axis was altered. Instead
of dropping exponentially, Shh::GFP ligand in Smo mutant neural tubes
displayed a linear profile across the ventral neural target field
(Fig. 3J). Thus, Smo, and
consequently Hh-responsiveness, is not required for Shh::GFP ligand to
concentrate in its normal apical domain but feedback mechanisms are likely to
prevent ligand from concentrating more generally on or around neural
progenitors. In addition, a negative-feedback mechanism restricts the range
and dictates the shape of the gradient consistent with the previous
description of Ptch1 and Hip1 feedback mutants
(Jeong and McMahon, 2005),
demonstrating that the target field gradient is clearly linked to Shh
morphogen activity.
|
; Kohtz et al.,
2001; Pepinsky et al.,
1998). Direct analysis of Shh::GFP in E8.75 Skn mutant
neural tubes (Fig. 3C,I,J)
demonstrated dramatically reduced levels of protein in target cells. A
significant accumulation of Shh::GFP ligand could only be detected in midline
neural progenitors that contact the notochord. Significantly, Shh::GFP ligand
was apically localized within these cells
(Fig. 3I). As Skn
mutants display a markedly different distribution from Smo mutants,
the reduction of concentrated Shh::GFP ligand is unlikely to simply reflect a
loss of responsiveness within the neural target field to non-palmitoylated Shh
ligand. More likely, this reflects a failure of cell-surface proteins to bind
or traffic non-palmitoylated Shh::GFP ligand in vivo. In addition, no obvious
difference in Shh::GFP production was observed in the notochord between each
class of mutant embryo. Thus, the observed changes in Shh::GFP ligand
distribution in Smo and Skn mutants provide strong genetic
evidence that the observed target field distribution is related to the
morphogen-based patterning activities of Shh. However, neither appropriate
lipid modification nor Shh responsiveness is absolutely required for Shh
ligand to accumulate at the apical region of cells in the target field.
|
).
From this, one might expect Shh ligand to quickly establish a long-range
gradient across the ventral neural tube so that target cells can acquire
positional information. We therefore examined the Shh::GFP ligand distribution
when each progenitor cell type first occupied the ventral midline: p2 (T1),
pMN (T2) or p3 (T3) in 6- to 12-somite-stage embryos
(Fig. 4A-F). To track changes
in the spatial distribution of ligand, the apical region of the ventral neural
tube was divided into three fixed regions (R1, R2 and R3). R3 covers the
apical region associated with ventral midline cells, whereas R2 and R1 cover
the apical region of more-dorsally positioned progenitors. When p2 progenitors
occupied the ventral midline (T1), Shh::GFP ligand was only detected in region
R3 (Fig. 4H). Shh::GFP ligand
increased in region R3 when the ventral midline was occupied by pMN
progenitors (T2) and expanded dorsally into region R2, where p2 progenitors
were now positioned (Fig. 4I).
When p3 occupied the ventral midline (T3), Shh::GFP ligand again increased in
region R3 and could now be detected in regions R2 and R1, where pMN and p2
progenitors reside, respectively (Fig.
4J). Thus, the target field gradient of Shh::GFP ligand, like the
Shh-dependent neural pattern, emerges over time, beginning at the apical
region of ventral midline cells and then spreading dorsally. It remains to be
determined whether the dorsal movement of Shh::GFP ligand reflects actual
ligand movement, or a dorsal expansion of ventral cells that retain Shh::GFP
ligand, or both.
Next, we quantified the accumulation of Shh::GFP ligand at the apical
region of ventral midline cells (R3) when each progenitor cell type first
appeared. We found that the level of Shh ligand progressively increased,
forming a temporal gradient at region R3 as ventral cell types emerged
(Fig. 4K). However, unlike the
exponential gradient spanning the three ventral cell types at T3
(Fig. 4L), the temporal
gradient is linear. Although it is not clear which gradient is relevant to Shh
morphogen activity, the temporal gradient is more consistent with the profile
of Shh concentrations that progressively specify more-ventral cell types in
vitro (Ericson et al.,
1997).
Shh::GFP ligand concentrates at the base of the primary cilium
Next we sought to identify the subcellular location where Shh::GFP ligand
concentrates in target cells. A number of studies have shown that Hh signaling
components, including Shh (Rohatgi et al.,
2007), can be found at the primary cilium of target cells,
although not in the context of active neural patterning
(Haycraft et al., 2005;
Huangfu and Anderson, 2005;
Rohatgi et al., 2007;
Scholey and Anderson, 2006;
Yoder, 2006). A primary cilium
is present on most mammalian cells and is an apical structure in epithelial
tissue. Staining for acetylated 
-tubulin, a marker for the shaft of the
primary cilium (Essner et al.,
2002), revealed a dense, complicated network of microtubules at
the apical region of the neural tube, preventing an unambiguous identification
of the primary cilium in this region (data not shown). The absence of other
cilial-specific markers prevented a direct analysis of the primary cilium
itself. At the cilial base, a centriole-containing basal body generates the
microtubule scaffold supporting the primary cilium, whereas the adjacent
transition zone acts as a loading dock for intraflagellar transport (IFT)
proteins that traffic cargo to the cilial compartment
(Rosenbaum and Witman, 2002;
Scholey, 2003). Gamma-tubulin
is a component of the basal body, whereas polaris (Ift88) demarcates the
transition zone (Haycraft et al.,
2005; Taulman et al.,
2001). As individual punctate structures can be resolved
(Fig. 5A and data not shown),
we were able to determine whether Shh::GFP ligand puncta preferentially
associate with these structures in the apical region of the ventral neural
tube. In addition, we focused our analysis on 8-somite-stage neural tubes as
the amount of Shh::GFP ligand in the apical region at later stages is too high
for co-localization analysis.
Strikingly, a three-way comparison of -tubulin, polaris and Shh::GFP
ligand puncta within the apical gradient showed that most (74%) of the
Shh::GFP ligand puncta associated (within 1 µm) with the basal body and
transition zone (Fig. 5A-C,J).
By contrast, only a small fraction (17%) of Shh::GFP puncta associated with
the lysosome, an organelle expected to degrade internalized Shh ligand
(Fig. 6A-C). To determine
whether there are regional differences in the association of Shh::GFP ligand
puncta with the basal body, we quantified the basal body association within
three volumes (V1, V2 and V3) along the D-V axis
(Fig. 7A). The Shh::GFP/basal
body association progressively decreased ventrally
(Fig. 7B), with all Shh::GFP
ligand puncta associated with the basal body at the most dorsal volume (V1)
and only 50% at the most ventral volume (V3). The change in association might
be due to differences in the nature of Shh signaling at different D-V
positions. Alternatively, there could be temporal differences. For example,
cells at the ventral midline might have been exposed to Shh signaling for
longer periods of time, allowing negative-feedback components to drive ligand
away from the basal body and towards the lysosome for degradation. Regardless,
our analysis indicates that the bulk of Shh::GFP ligand within the target
field gradient is associated with the apically positioned basal body of neural
progenitors during active Shh morphogen-based patterning of the ventral neural
tube, a highly asymmetric distribution within target cells. As Shh::GFP ligand
can localize to this position in Smo and Skn mutants
(Fig. 5D-I), secondary
response-dependent processes are not required or responsible for this specific
intracellular distribution.
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-tubulin
revealed an extensive microtubule network that spans the apical-to-basal
surfaces of ventral midline cells (Fig.
6D). Strings of small Shh::GFP ligand puncta (smaller than the
basal-body-associated accumulations) were found in close association with
these microtubules (Fig. 6E,F).
As this association occurs at stages prior to Shh::gfp expression at
the ventral midline and in Gli2 mutant neural tubes
(Fig. 6D-F), the small Shh
ligand puncta must have originated from the notochord. Thus, these puncta
might represent Shh::GFP ligand trafficking from the Shh::GFP-expressing
notochord into the neural target field, a process that might be facilitated by
the physical contact between these two distinct tissues. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Clearly, understanding how Shh is distributed as ventral cell fates emerge
is crucial to our understanding of this process. To address this question, we
replaced the endogenous Shh protein with a fluorescently tagged form in the
mouse and visualized its distribution in the context of active neural
patterning in vivo. Shh ligand is first detected at the apical region of
ventral midline cells when patterning begins and then extends dorsally as
neural patterning progresses (Fig.
4). Thus, the Shh ligand profile in the ventral neural target
field does not appear to rapidly reach a steady state, but appears to slowly
emerge with the ventral pattern. That the observed target field accumulation
is related to Shh morphogen action is supported by the sensitivity of the
gradient profile to both negative feedback and Skn-mediated palmitoylation,
both of which have been shown to be crucial modulators of Shh morphogen
activity in the neural tube (Briscoe et
al., 2001; Chen et al.,
2004; Jeong and McMahon,
2005).
|
2- to 3-fold
increase between distinct ventral cell identities
(Ericson et al., 1997). These
data suggest that Shh morphogen activity might be distributed temporally at
the ventral midline during neural tube patterning, consistent with a
time-based model for Shh-dependent neural patterning
(Stamataki et al., 2005). In
this model, a specific cell identity may be specified at a precise ventral
location where signaling is progressively increasing. However, as cell
descendants assume more-dorsal positions in this rapidly dividing tissue, they
now enter a new zone where Shh levels may actually decrease to a level
insufficient to promote further specification. The relationship between time
spent within a specific concentration range of Shh ligand and the locking in
of a specific cell fate remains unclear. However, experiments aimed at
addressing this might shed light on the in vivo patterning process.
The cilium base and Shh signal transduction
The localization of Shh ligand adjacent to the basal body of target cells
is provocative in light of other studies that suggest that the primary cilium
plays a role in Hh pathway activation. The primary cilium is found on most
cells and is an apical, membrane-bound projection supported by a microtubule
scaffold generated by the centriole-containing basal body
(Rosenbaum and Witman, 2002).
Plus-end (distal) -directed kinesins and minus-end (proximal) -directed
dyneins transport cargoes required for the formation, maintenance and function
of the primary cilium on its microtubule scaffold. In this, IFT proteins
directly bind and traffic these cargoes through the cilial compartment.
Ablating IFT genes in the mouse disrupts primary cilium formation and
significantly reduces Hh signaling in the neural tube
(Huangfu et al., 2003).
Genetic epistasis analysis suggests a role for IFT in Gli activity
(Haycraft et al., 2005;
Huangfu and Anderson, 2005;
Liu et al., 2005). Further,
cell biological studies suggest that Smo and Ptch1 activity might be
restricted to the primary cilium of target cells
(Corbit et al., 2005;
Rohatgi et al., 2007).
How does the observed accumulation of Shh ligand at the cilium base of
target cells fit with the current view of cilium function in Shh signaling?
Ptch1 antagonizes Smo in the absence of ligand and Ptch1 appears to be present
at the base and within the shaft of the primary cilium
(Rohatgi et al., 2007). As Shh
can be detected in the shaft when Ptch1::YFP is overexpressed in
Ptch1 mutant MEF cells, it has been proposed that Shh binds and
thereby promotes the disappearance of Ptch1 from the primary cilium. The
disappearance of Ptch1 is associated with the accumulation of Smo on the
primary cilium and with active signaling
(Corbit et al., 2005;
Rohatgi et al., 2007),
although the movement of Ptch1 off the primary cilium is not essential for Smo
to function (Rohatgi et al.,
2007). Given these findings, it is perhaps surprising that Shh
ligand accumulates at the cilium base of neural progenitors during active Hh
signaling. However, it is notable that Ptch1 is found predominantly at the
cilium base in vivo (Rohatgi et al.,
2007). Here, Ptch1 would be well positioned to regulate Smo
transport to the primary cilium. Thus, it is possible that Shh ligand traffics
to the cilial base and its interaction there with Ptch1 might permit the
transport of Smo onto the shaft of the primary cilium to activate the Hh
pathway. Reagents that enable the dynamic visualization and spatiotemporal
quantification of these crucial signaling components in their physiological
contexts will greatly help in understanding these mechanisms. The
Shh::gfp allele represents the first step in this direction.
Shh target field trafficking
The observed apical accumulation of notochord-derived Shh ligand appears to
occur by a Shh-specific mechanism, as notochord-derived Wnt1 ligand has a
distinct distribution (Fig.
2L). Several cell-surface, Shh-binding proteins are expressed in
the ventral neural tube and may be localized and bound to Shh within the
apical compartment of neural progenitors. As the apical gradient of ligand
accumulation persists in Smo mutant neural tubes, where all Hh
signaling is lost, the responsible ligand-binding protein must be present
during the primary response of the neural target field to Shh ligand. Several
Shh-signal-promoting binding partners have been identified in addition to the
primary receptor, Ptch1 (e.g. Gas1, Cdon and Boc)
(Allen et al., 2007;
Tenzen et al., 2006). Notably,
we observe a dramatic reduction in the apical accumulation of
non-palmitoylated Shh ligand. As palmitoylation is required for Shh ligand
activity, its loss may lead to a reduced association with these binding
partners. That Ptch1 accumulates at the base of the primary cilium is
consistent with this view (Rohatgi et al.,
2007). However, Ptch1 levels are themselves directly related to
Shh signaling so there might not be a simple quantitative association between
Ptch1 and Shh puncta. We have been unable to directly visualize Ptch1 with
available antibodies. Also, the rapid activation of Shh expression
within neural target cells following loss of Ptch1 activity
(Goodrich et al., 1997) (data
not shown) prevents notochord-derived Shh::GFP from being visualized in a
Ptch1 mutant background.
How does Shh ligand move into and through the neural target field? Our
observations of Shh ligand puncta on stabilized microtubules that span ventral
neural progenitors from notochordal to apical surfaces indicate that Shh might
be endocytosed and subsequently trafficked through the cell to its site of
apical accumulation. Trafficking of protein from the basolateral to apical
surfaces of epithelial cells, or transcytosis, occurs in several physiological
contexts (Tuma and Hubbard,
2003) and would provide an elegant method of regulating the
distribution of Shh signals.
At present, the resolution of microscopy does not enable us to unambiguously determine whether these smaller puncta are in, on, or between cells. Further, the dorsal extension of Shh ligand distribution within the neural target field could occur by a similar mechanism to that regulating uptake in the ventral midline cells, through the apical release of Shh ligand into the lumen of the neural tube, or through the cell division and growth of ventral midline cells such that daughter cells carry with them Shh ligand acquired at a more ventral position. Resolving the mechanisms that contribute to the temporal gradient of Shh ligand will require dynamic approaches to visualize morphogen action.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/6/1097/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: 1550 4th St., Room 282, UCSF Mission Bay campus, San
Francisco, CA 94143, USA
Present address: Department of Surgery/Urology, Children's Hospital Boston,
Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
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