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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02850
1 Institute for Cellular and Molecular Biology, University of Texas at Austin,
Austin, TX 78712-0248, USA.
2 Section of Neurobiology and University of Texas at Austin, Austin, TX
78712-0248, USA.
3 Institute for Neuroscience, University of Texas at Austin, Austin, TX
78712-0248, USA.
Author for correspondence (e-mail:
agarwala{at}mail.utexas.edu)
Accepted 13 March 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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loop2,
we show that HH signaling is necessary and can act directly at a distance to
specify midbrain cell fates. Ventral midbrain progenitors extinguish their
dependence upon HH in a spatiotemporally complex manner, completing cell-fate
specification at the periphery by Hamburger and Hamilton stage 13. Thus,
patterning at the lateral periphery of the ventral midbrain is accomplished
early, when the midbrain is small and the HH signal needs to travel relatively
short distances (approximately 30 cell diameters). Interestingly, single-cell
injections demonstrate that patterning in the midbrain occurs within the
context of cortex-like radial columns of cells that can share HH blockade and
are cytoplasmically connected by gap junctions. HH blockade results in
increased cell scatter, disrupting the spatial coherence of the midbrain arc
pattern. Finally, HH signaling is required for the integrity and the signaling
properties of the boundaries of the midbrain (e.g. the midbrain-hindbrain
boundary, the dorsoventral boundary), its perturbations resulting in abnormal
cell mixing across `leaky' borders.
Key words: Midbrain-hindbrain boundary, Motor and dopaminergic neurons, Morphogen, Cell affinities, Size regulation, Midbrain radial columns, Chick
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the case of the midbrain, the identity of the ventral
midbrain or rostral floor plate (rFP) as a signaling center is firmly
established (Agarwala et al.,
2001; Blaess et al.,
2006; Fedtsova and Turner,
2001). The rFP occupies the ventral midline of the midbrain and
secretes the signaling molecule sonic hedgehog (SHH), whose role in pattern
formation is the focus of intense study
(Ingham and McMahon,
2001).
Hedgehog (HH) signal transduction begins with HH binding to its receptor
and negative regulator, PTC1 (Hooper and
Scott, 2005; Ingham and
McMahon, 2001; Marigo et al.,
1996; Stone et al.,
1996). In the absence of HH signaling, PTC1 maintains a
constitutive block on the transmembrane protein smoothened (SMO) so that no
signaling can occur (Akiyama et al.,
1997; Alcedo et al.,
1996). New findings suggest that, in the absence of the ligand,
PTC1 can induce provitamin D3, which binds SMO in adjacent cells to block HH
activation (Bijlsma et al.,
2006). In the presence of HH, the PTC1-mediated block on SMO is
lifted. HH signaling then occurs via a complex cascade, which eventually
converges upon the activator-(GLI1, GLI2, GLI3) or repressor-(chiefly GLI3)
function of the GLI/Ci family of transcription factors
(Aza-Blanc et al., 1997;
Bai et al., 2004;
Dai et al., 1999;
Litingtung and Chiang, 2000;
Sasaki et al., 1999;
Wijgerde et al., 2002).
Among vertebrates, one of the best-understood examples of the role of HH in
patterning is in the ventral spinal cord
(Jessell, 2000). Gain- and
loss-of-function studies have shown that HH is both necessary and sufficient
for cell-fate specification in the spinal cord
(Briscoe and Ericson, 2001;
Chiang et al., 1996;
Zhang et al., 2001). HH is
directly required for cell-fate specification and can pattern cell fates at
long range (approximately 15-20 cell diameters)
(Briscoe et al., 2001;
Wijgerde et al., 2002).
A role for HH signaling in the regulation of cell affinities has been found
in the fly wing imaginal disc and abdominal ectoderm
(Blair and Ralston, 1997;
Lawrence et al., 1999;
Rodriguez and Basler, 1997).
In each tissue, differential HH signaling creates two compartments that
display distinct and inheritable affinities. Thus, cells of a compartment and
their lineal relatives cohere with each other and do not intermix with those
of the other compartment. As a result, the compartments become separated by a
sharp, lineage restriction boundary exhibiting signaling properties
(Blair, 1992;
Garcia-Bellido et al., 1973;
Lawrence et al., 1999;
Morata and Lawrence, 1975).
These results implicate HH signaling in the establishment of tissue boundaries
and in the maintenance of a spatially coherent pattern
(Dahmann and Basler, 1999). A
loss of spatial organization has also been reported in several HH-pathway
mutants in mouse (Shh-/-;Gli3-/-,
Smo-/-;Gli3-/-,
Gli2-/-;Gli3-/-) and chick (e.g. the
talpid2 mutant)
(Agarwala et al., 2005;
Bai et al., 2004;
Litingtung and Chiang, 2000;
Wijgerde et al., 2002).
Recently, HH signaling has also been implicated in the maintenance of
orthogonal signaling centers in the vertebrate limb and in the
midbrain-hindbrain boundary (MHB) of the neural tube
(Aoto et al., 2002;
Blaess et al., 2006;
Khokha et al., 2003). However,
whether the regulation of boundaries is a general feature of HH action among
vertebrates is not yet known.
In this study, we analyzed the role of HH signaling in the chick midbrain,
where stripes of cell fates (midbrain arcs) develop parallel to the rFP source
of SHH (Agarwala et al.,
2001; Sanders et al.,
2002). In vivo misexpression studies have shown that ectopic SHH
can recapitulate the entire midbrain pattern of cell fates in a
concentration-dependent manner (Agarwala
and Ragsdale, 2002; Agarwala et
al., 2001). No ventral cell fates remain in the
Shh-/- mouse midbrain by embryonic day (E)11.5, when the
entire midbrain exhibits a dorsal phenotype
(Blaess et al., 2006;
Fedtsova and Turner, 2001).
Although these studies demonstrate the importance of SHH in the developing
midbrain, they do not permit a precise cellular and molecular analysis of the
role of HH signaling in establishing midbrain pattern. Nor do they elucidate
the physical nature of the HH signal; for example, its range (short or long),
mode (direct or indirect), timing or duration of action.
To address these issues, we perturbed HH function in the ventral midbrain
by in vivo misexpression of
Ptc1loop2, a mutated form of
PTC1 that has been used previously to successfully block HH signaling
(Briscoe et al., 2001;
Kiecker and Lumsden, 2004). We
show that HH is directly required for cell-fate specification within columns
of midbrain cells, which are cytoplasmically connected and likely to be
clonally related (Noctor et al.,
2001). HH signaling acts at long range (approximately 31 cell
diameters) at Hamburger and Hamilton (H&H) stage 13, when cell-fate
specification is complete at the lateral periphery of the ventral midbrain
(Hamburger and Hamilton,
1951). Beyond this time, continued dependence upon HH is only seen
within lateral regions of the rFP and cell fates associated with it. Our
results also suggest that the blockade of HH signaling increases cell
proliferation and inhibits differentiation within the midbrain. Finally, HH is
required for the spatial organization of midbrain cell types and for the
maintenance of the boundaries of the midbrain. Perturbations of HH signaling
thus result in the admixture of midbrain cells with each other and with cells
from juxtaposed tissues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Expression vectors
Embryos were electroporated with either enhanced green fluorescent protein
(EGFP; EFX-EGFP),
Ptc1loop2
(pCIG-Ptc1loop2) or
SHH (XEX-SHH)-containing expression vectors. The construction of
Ptc1loop2 and XEX-SHH has been
described previously (Agarwala and
Ragsdale, 2002; Agarwala et
al., 2001; Briscoe et al.,
2001). The second large extracellular loop of mouse Ptc1
(also known as Ptch1 - Mouse Genome Informatics) (corresponding to
amino acids 793-998), which normally binds the HH ligand, has been deleted in
the Ptc1loop2 construct.
Ptc1loop2 can thus maintain a
constitutive blockade on SMO, acting as a dominant-negative regulator of HH
signaling (Briscoe et al.,
2001). The EFX-EGFP construct was created by ligating the
BamHI-NotI fragment (800 bp) of pEGFPN1 (Clontech) into the
plasmid EFX3C (Agarwala et al.,
2001).
In ovo electroporation
DNA (1-3 µg/µl) was electroporated into H&H stages 6-20 embryos
according to previously established protocols
(Agarwala and Ragsdale, 2002;
Agarwala et al., 2001;
Momose et al., 1999).
Electroporated embryos were returned to the incubator for 1-7 days prior to
collection for further analyses. Only 20% of the embryos electroporated
between H&H stages 6-8 survived to E5.
In situ hybridization
Embryos were harvested between E3 and E8, and were then immersion-fixed in
4% paraformaldehyde. Digoxigenin (DIG)- or Fluorescein-conjugated antisense
riboprobes were prepared from cDNAs for class III ß-tubulin, cyclin B2,
cyclin D1, EVX1, FOXA2, GLI2, FGF8, ISL1, LMX1B, NKX2.2, OTX2, PAX6, PAX7,
PHOX2A, PTC1, SERRATE1, SHH, TH and WNT1, and from mouse
Ptc1. The antisense riboprobe for EGFP was generated from pBS-EGFP,
constructed by subcloning the BamHI-NotI fragment of pEGFPN1
(Clontech) into the bluescript plasmid (Stratagene). One- or two-color
whole-mount in situ hybridizations were conducted according to published
protocols (Agarwala and Ragsdale,
2002; Agarwala et al.,
2001).
Cell-death assay
Whole-mount cell-death assays were carried out on E5 embryos using
previously published protocols (Agarwala et
al., 2005; Yamamoto and
Henderson, 1999) (see also T. A. Sanders, PhD thesis, University
of Chicago, 2001).
Midbrain explants
For explant cultures, embryos were electroporated as usual with EFX-EGFP or
pCIG-Ptc1loop2. Midbrain
explants were prepared as previously published with either: (a) an attached
dorsal midbrain; (b) no dorsal midbrain; or (c) no dorsal mid- or hind-brain
(Agarwala and Ragsdale, 2002).
Prepared explants were cultured for 3 days prior to harvesting.
Bromodeoxyuridine labeling
Bromodeoxyuridine [BrdU; 1 µl; 15 mg/ml (50 µM) in PBS; Sigma] was
intravenously injected into E5 embryos electroporated at H&H stage 10.
Injected embryos were incubated for 30 minutes before fixation. In situ
hybridization and the detection of BrdU labeling were combined according to
established protocols (Agarwala et al.,
2001) (see also T. A. Sanders, PhD thesis, University of Chicago,
2001).
Whole-cell current-clamp recordings
Embryos were explanted at H&H stage 10 as described above and neuronal
progenitors were visualized using infrared DIC microscopy (Zeiss Axioscope 2)
and a Dage-MTI Newvicon tube camera. Whole-cell current-clamp recordings were
made at room temperature using somatic patch pipettes with open tip
resistances of 2-4 M
. Alexa-Fluor 488 (30 µM) was added to the
internal solution, which was made according to published protocols
(Scott et al., 2005).
Dye-coupled cells were identified by visualizing Alexa-Fluor 488 with
fluorescence microscopy (EXFO X-cite 120 light source, Photometrics Cascade
512B camera).
Orientation of photomicrographs
Unless mentioned, images of unilaterally electroporated E5 embryos are
presented as whole mounts with rostral to the top and the ventricular surface
facing the viewer (open-book view). The electroporated side is presented to
the right, the left side serving as a control. Where crucial, the age of
electroporation (Fig. 4) or the
age of harvest (remaining figures) is provided on the photomicrographs.
Embryos bilaterally electroporated with
Ptc1loop2 are identified with
`bi' on respective panels and EGFP-electroporated controls are provided for
comparison. Sections are shown with the ventricular surface at the top and the
pial surface at the bottom.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These
are marked by the gene expression of PHOX2A and tyrosine hydroxylase
(TH) in the most medial arc (arc 1), and more laterally (at a
distance from the SHH source) by the expression of NKX2.2,
PAX6 and EVX1 (Fig.
1A,F,G and data not shown)
(Agarwala and Ragsdale, 2002).
We determined that the SHH source and the midbrain arc pattern were
not perturbed by control electroporations of EGFP
(Fig. 1A). The rFP markers
SHH and FOXA2 (HNF3ß) are transcriptional
targets of HH signaling in the midbrain
(Agarwala et al., 2001), and
were suppressed by Ptc1loop2
electroporations (Fig. 1B;
Fig. 4D).
Ptc1loop2 misexpression also
prevented the correct specification of all ventral midbrain cell fates,
resulting in their re-specification to more dorsal (e.g. PAX7+) fates
(Fig. 1C,
Fig. 4G,H,
Fig. 6A). We noted a
suppression of the PHOX2A+ oculomotor neurons, midbrain dopaminergic
(TH+) neurons, as well as the territories (NKX2.2+, PAX6+,
EVX1+) specified at a distance from the SHH source
(Fig. 1D-G and data not shown).
Taken together with our previous work, these results suggest that HH signaling
is both necessary and sufficient for cell-fate specification in the ventral
midbrain and can act directly at a distance to specify midbrain cell fates
(Agarwala and Ragsdale, 2002;
Agarwala et al., 2001).
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;
Lawrence et al., 1999;
Rodriguez and Basler, 1997).
An increased spread of cells was also noted within the ventral midbrain
following Ptc1loop2
electroporations (compare Fig. 1D and
1E; Fig. 1E-J).
This increased scatter was non-autonomous (e.g.
Fig. 1E,H), multidirectional,
increased dramatically over time (Fig. 1,
compare I,J with D,E) and affected progenitors as well as
differentiated neurons (see Fig. S1 in the supplementary material). As a
result of this scatter, a spatially coherent midbrain arc pattern could not be
formed following Ptc1loop2
electroporations (Bai et al.,
2004; Blair and Ralston,
1997; Lawrence et al.,
1999; Wijgerde et al.,
2002).
HH signaling inhibits proliferation and induces neuronal differentiation in the midbrain
HH signaling is known to accelerate progression through the cell cycle in
many model systems (Duman-Scheel et al.,
2002; Kenney and Rowitch,
2000; Roy and Ingham,
2002). By contrast, we found that the expression of known cell
cycle targets of HH signaling (cyclin B2, a marker of G2/M transition; and
cyclin D1, a marker of G1/S transition), as well as BrdU labeling (marking the
S phase of the cell cycle) all indicated greatly increased numbers of neuronal
progenitors following Ptc1loop2
electroporation (Fig. 2A-D)
(Masai et al., 2005).
Concomitant to the increased proliferation was a reduction in the number of
differentiated neurons demonstrated by the reduced thickness of the mantle
layer (Fig. 2C,D, double-headed
arrow) and reduced class III ß-tubulin expression
(Fig. 2E, inset). TUNEL
labeling indicated no significant differences in cell death between
Ptc1loop2 and
EGFP-electroporated midbrains (see Figs S2 and S3 in the
supplementary material).
To discount the possibility that the altered midbrain proliferation and
differentiation was due to a peculiarity of the
Ptc1loop2 construct itself, we
misexpressed SHH and found that cyclin D1 mRNA was severely reduced
in both the ventral and dorsal midbrain
(Fig. 2F,G and see Fig. S4 in
the supplementary material) (Guerrero and
Ruiz i Altaba, 2003; Thibert
et al., 2003). Finally, we compared the total size of midbrains
electroporated at H&H stage 9 with either SHH or
Ptc1loop2 and found that the
SHH, but not the
Ptc1loop2 electroporated
midbrains displayed a massive (>50%, in some cases) reduction in size
(Fig. 2H). Taken together,
these results are consistent with a role for HH signaling in the midbrain in
suppressing proliferation and inducing differentiation
(Bai et al., 2004;
Masai et al., 2005;
Wijgerde et al., 2002).
HH blockade reveals a cortex-like radial organization of the ventral midbrain neurepithelium
Following HH blockade, the expression of appropriate midbrain cell-fate
determinants (e.g. FOXA2, PHOX2A) was not only blocked
cell-autonomously within cells expressing the
Ptc1loop2 (mouse Ptc1)
transgene, but also in `haloes' immediately surrounding the
Ptc1loop2+ cells
(Fig. 3A;
Fig. 1B,E). In E5
cross-sections, these `haloes' (cells that did not express spatially
appropriate HH-target fates despite appearing
Ptc1loop2 negative), were
organized into `columns' of cells that spanned the ventricularpial (radial)
axis and were radially aligned with pially located
Ptc1loop2+ cells
(Fig. 3B).
|
|
; Kriegstein
and Noctor, 2004). We next determined whether midbrain columns were the result of HH blockade or a normal feature of midbrain organization. For this purpose, we shifted our analysis to E4, when the midbrain neurepithelium is predominantly composed of undifferentiated precursors and HH-blockade-mediated perturbation of proliferation and differentiation does not add additional complexity (Fig. 2A-E).
Columns of electroporated cells spanning the ventricular-pial axis could be
seen in
Ptc1loop2-electroporated
embryos at E4 (Fig. 3C). A
similar columnar organization was seen in midbrains electroporated with low
concentrations of EGFP (0.2 µg/µl) to yield only a few isolated
EGFP+ cells per brain (Fig.
3D,E). Thus, HH blockade neither induced nor disrupted the
columnar organization of the ventral midbrain. Notably, the EGFP+ cells
displayed the characteristic morphology of radial glial/neuronal precursors
(bipolar cells spanning the midbrain ventricular-pial axis and exhibiting
apical and basal processes with end-feet)
(Fig. 3E and data not shown)
(Malatesta et al., 2003;
Noctor et al., 2001).
Furthermore, when multiple EGFP+ cells were present within a single midbrain
column, they were cytoplasmically connected
(Fig. 3D, arrowhead).
Cytoplasmic connections (via gap junctions) among clonal relatives are a
feature of cortical columns and have been detected in dye-coupling experiments
(Noctor et al., 2001). Indeed,
single-cell injections in midbrain explants at H&H stage 10 with
Alexa-Fluor 488 (which crosses gap junctions, but does not diffuse across cell
membranes) resulted in the instantaneous labeling of up to three cells,
demonstrating the presence of gap junctions among midbrain progenitors
(n=5; Fig. 3F).
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loop2 negative cells that
are radially associated with
Ptc1loop2+ cells are unable to
express appropriate HH-target fates because they divide and differentiate
under reduced HH conditions. Such conditions could be created by the
cytoplasmic inheritance of low/undetectable levels of
Ptc1loop2 (cell-autonomous) or
due to the transfer of small inhibitory molecules (e.g. provitamin D3) among
neuronal precursors via gap junctions
(Bijlsma et al., 2006). For
precision, we have described the radial effects of
Ptc1loop2 electroporations as
being `radially associated' or `associated' with
Ptc1loop2+ cells, rather than
being cell-autonomous or non-autonomous.
Spatiotemporal regulation of ventral midbrain patterning by HH
We next determined the spatiotemporal sequence in which midbrain cell fates
extinguished their dependence upon HH signaling. Compared with
EGFP-electroporated controls (Fig.
4A, Fig. 5A), very
few Ptc1loop2+ cells were seen
within the medial region of the rFP in bilateral electroporations
(Fig. 4B,F,
Fig. 5B)
(Briscoe et al., 2001;
Wijgerde et al., 2002). When
they did appear at the midline, they could only suppress SHH or
FOXA2 gene expression along the caudal (near the MHB), but not
anterior (Fig. 4B, arrowhead),
midline of rFP between H&H stages 6-11
(Fig. 4B,C,
Fig. 7A). By sharp contrast,
floor plate markers (SHH, FOXA2) could be blocked in lateral regions
of the rFP in electroporations conducted between H&H stages 15-20
(Fig. 4D,
Fig. 7A-C).
|
loop2 misexpression between
H&H stages 8-13 (n>40) resulted in the uniform blockade of all
arc-specific cell fates throughout the mediolateral (ML) axis of the ventral
midbrain (Fig. 4E,F). Although
ML differences in specification were not noticed across the midbrain arcs
following HH blockade during this time, cell-fate specification was more
severely affected near the MHB compared with more rostral regions,
particularly within the medial arc territory (7/10 embryos;
Fig. 4F).
In electroporations beyond H&H stage 13, only cell fates associated
with the lateral regions of the rFP and arc 2 (the region between the
PHOX2A and PAX6 territories) were affected by HH blockade
(Fig. 4G,H and see Fig. S5 in
the supplementary material). Intriguingly, this region was marked by the
ectopic presence of more lateral (e.g. PAX6+) phenotypes occurring
both non-autonomously (Fig. 4H,
arrowhead) and in radial association with misexpressed
Ptc1loop2
(Fig. 4H, arrow). We saw a
similar mixed phenotype (radially associated and non-autonomous) throughout
the study and interpret these results as a combination of re-specified cell
fates to a more dorsal identity (radially associated with
Ptc1loop2+ cells) and abnormal
cell scatter (non-autonomous; see Discussion).
Our data suggest that the anterior midline rFP was not affected by our
manipulations between H&H stages 6 and 20, possibly because they are
specified earlier or independent of HH signaling
(Patten et al., 2003).
HH-mediated specification of the remaining ventral midbrain cell fates occurs
in at least three temporal phases (Fig.
7A-C). First, prior to H&H stage 11, the caudo-medial region
of the rFP becomes independent of HH signaling (step 1;
Fig. 7A). This is followed by
most ventral midbrain cell fates becoming independent of HH signaling by
H&H stage 13 (step 2; Fig.
7B). Beyond H&H stage 13, only the lateral regions of the rFP
and cells associated with it exhibit a dependence upon HH signaling and
continue to do so at least until H&H stages 17-20 (step 3;
Fig. 7C).
Perturbations of HH signaling result in a disruption of midbrain boundaries
In the fly wing and abdomen, HH perturbations result in a disruption of
cell affinities, evident as a spatially disorganized pattern and disrupted
compartment boundaries (Fig. 1)
(Lawrence et al., 1999). We
asked whether midbrain boundary perturbation accompanied the disruption of
spatial pattern as well (Aoto et al.,
2002; Blaess et al.,
2006; Lawrence et al.,
1999; Zervas et al.,
2005).
The midbrain-hindbrain boundary
Ptc1loop2 misexpression
resulted in a broadening of the MHB and a non-autonomous scattering of
WNT1+ cells that was not seen in control brains
(Fig. 5A,B). Strikingly,
Ptc1loop2 manipulations
resulted in the intermingling of midbrain (OTX2+) and MHB/hindbrain
cells (FGF8+; Fig.
5C,D and see Fig. S6 in the supplementary material). This was
accompanied by a dramatic broadening of the FGF8+ MHB territory
(Fig. 5C,D). The broadening
could not be explained by a repression of OTX2, an expansion of
GBX2 or the ectopic presence of mis-specified cells
(Fig. 5D, Fig. S6 in the
supplementary material and data not shown). Instead, the broadening could be
attributed to enhanced cell proliferation within the MHB, as demonstrated by
the dramatic increase of cyclin D1+/FGF8+ cells
(Fig. 5E,F). Thus, reduced HH
signaling results in an enlarged MHB that is not sharply defined and across
which cell-mixing can occur (Vaage,
1969; Zervas et al.,
2004).
The dorsoventral boundary
The disruption of the MHB following
Ptc1loop2 manipulations
prompted us to examine the dorsoventral (DV) boundary. When electroporated
with Ptc1loop2, ectopic
PAX7+ cells, normally confined to the dorsal midbrain, were noticed
in the ventral midbrain (Fig.
6A). We also observed that the expression of the DELTA homolog,
serrate 1, was disrupted along the DV boundary following
Ptc1loop2 electroporations
(Fig. 6B).
The presence of PAX7+ cells in the ventral midbrain could result
from a conversion of ventral midbrain cells to a dorsal fate or from the
movement of dorsal cells into the ventral midbrain because of a breach in the
signals that normally restrict their admixture. To distinguish between these
possibilities, we resorted to an explant system, in which all PAX7+
dorsal tissue could be removed prior to electroporation with
Ptc1loop2
(Agarwala and Ragsdale, 2002).
In EGFP-electroporated control explants with or without an intact
tectum, no PAX7+ cells were ever seen in the ventral midbrain
(n=11/11; Fig. 6C and
data not shown). When explants prepared without any associated PAX7+
tissue (dorsal midbrain and hindbrain; n=4/4) were electroporated
with Ptc1loop2, PAX7+ cells
could be observed within the ventral midbrain, suggesting that some ventral
midbrain cells were converted to a dorsal (PAX7+) phenotype in the
absence of HH signaling (Fig.
6D).
|
loop2 near the DV boundary
followed by the simultaneous detection of PAX7 and the
Ptc1loop2 transgene. Ectopic
PAX7+ cells were not seen near the DV boundary in
EGFP-electroporated brains (n=0/5;
Fig. 6E). However, there was
always a small number of cells that displayed PAX7 expression
non-autonomously in
Ptc1loop2-electroporated brains
(n=7/7; Fig. 6F).
Taken together, our results are consistent with both a transformation of
ventral midbrain cell fates to dorsal fates and with a non-autonomous movement
of dorsal cells into the ventral midbrain due to an MHB-like disruption of the
DV boundary. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The specification of HH-target midbrain cell fates is
largely complete by H&H stage 13, with a continued requirement for HH
signaling beyond this time point only in lateral regions of the rFP and
associated cell types (e.g. arc 2; Fig.
7A-C). Interestingly,
Ptc1loop2 electroporations
result in increased cell proliferation and reduced differentiation, closely
resembling the size regulation in
Gli2-/-;Gli3-/- and
Smo-/-;Gli3-/-, but not
Shh-/-, mice (Fig.
7E) (Bai et al.,
2004; Litingtung and Chiang,
2000; Wijgerde et al.,
2002). Finally, HH signaling is required for the correct spatial
patterning of midbrain cell types and for the integrity of the boundaries of
the midbrain (MHB, DV boundary; Fig.
7D).
The range of HH action in the midbrain
We determined that direct HH signaling was required at the lateral edge of
the ventral midbrain and that this requirement was extinguished by H&H
stage 13 (Figs 1,
4). The restriction of
PAX7 expression to the dorsal midbrain by HH is a measure of the
range of HH signaling (Ericson et al.,
1996; Wijgerde et al.,
2002). The distance between the lateral limit of the SHH
source and the ventral limit of the PAX7 domain in the midbrain at
H&H stage 10, when midbrain patterning is ongoing, is approximately 180
µm. Based on our dye-coupling experiments
(Fig. 3F), the average cell
diameter of midbrain neurepithelial cells at H&H stage 10 is approximately
7.5 µm (range 5-10 µm; data not shown). Thus, at H&H stage 10, the
SHH signal must travel up to approximately 24 cell diameters to influence cell
fates at the lateral periphery of the ventral midbrain. This distance
increases to approximately 31 cell diameters at H&H stage 13, which is
only 1.5 times the distance of 12-20 cell diameters traversed by the HH signal
in the fly wing, vertebrate limb and spinal cord
(Briscoe et al., 2001;
Ericson et al., 1996;
Harfe et al., 2004;
Tabata and Takei, 2004;
Wijgerde et al., 2002). Thus,
despite the ultimately different sizes of the midbrain and spinal cord, the
problem of getting the HH signal across long distances is circumvented by
accomplishing midbrain cell-fate specification relatively early, when the
midbrain size is small and comparable to the spinal cord. The role of
continued SHH expression beyond this time point is not known,
although cell survival, axon guidance, dorsal patterning and size regulation
are possible functions (Blaess et al.,
2006; Ishibashi and McMahon,
2002).
HH signaling regulates cell cycle and differentiation in the developing midbrain
Blockade and overexpression experiments demonstrate that HH regulates
midbrain size by preventing cell proliferation and by inducing differentiation
with no significant alterations in cell survival
(Fig. 2). Although midbrain
size regulation in the chick midbrain following
Ptc1loop2 manipulations differs
from that reported for the Shh-/- mouse, it strongly
resembles the phenotype of the mouse
Gli2-/-;Gli3-/- and
Smo-/-;Gli3-/- spinal cords, in which
no HH signaling is possible (Bai et al.,
2004; Blaess et al.,
2006; Chiang et al.,
1996; Ishibashi and McMahon,
2002; Wijgerde et al.,
2002). Why size regulation differs between these two sets of mice
is not clear, but may depend upon the levels of GLI repressor present in each
manipulation (Cayuso et al.,
2006) and also upon the ligand-independent interactions between
the cell cycle and HH pathway members
(Barnes et al., 2005).
Interestingly, HH signaling in the retina and cerebellar granule cells
regulates multiple aspects of proliferation and differentiation (e.g. G1-S
transition, cell-cycle exit and neuronal differentiation)
(Duman-Scheel et al., 2002;
Pons et al., 2001;
Wechsler-Reya and Scott,
1999). Thus, whether HH is a positive or a negative regulator of
size may depend upon the cellular context and the level of the HH signaling
cascade at which a given HH perturbation is targeted
(Masai et al., 2005;
Neumann, 2005).
HH blockade results in increased cell scatter and disrupts the midbrain arc pattern
Increased cell scatter and a disruption of the arc pattern followed
Ptc1loop2 electroporation in
the ventral midbrain (Fig. 1,
Fig. 4G,H). Similar disruptions
in spatial patterning have also been seen following HH perturbations in
multiple systems in the fly, mouse and chick
(Agarwala et al., 2005;
Bai et al., 2004;
Lawrence, 1997;
Litingtung and Chiang, 2000;
Wijgerde et al., 2002). In the
chick midbrain, spatially inappropriate cell fates appeared both in radial
association with Ptc1loop2+
cells as well as non-autonomously (e.g.
Fig. 1H,
Fig. 4G,H). Because robust
Ptc1loop2 transgene was seen at
E5-E6 (e.g. Fig. 1B,
Fig. 4G), the selective
shutdown of transgene expression in subgroups of manipulated cells is an
unlikely explanation for the dual phenotype. We noticed that
cell-mixing/movement across midbrain boundaries (MHB, DV boundary) following
HH blockade invariably occurred in a non-autonomous manner (Figs
5,
6). Thus, a possible
explanation for this dual phenotype is that it represents a combination of
cell-spread (non-autonomous) and cell-fate re-specification (in radial
association with Ptc1loop2+
cells).
Previous studies have noted a cell-autonomous, stepwise dorsalization of
cell fates and a non-autonomous, stepwise dorsal-to-ventral transformation of
cell fates due to a failure of
Ptc1loop2+cells to sequester HH
(Briscoe et al., 2001).
However, in the midbrain, the non-autonomous effects were non-directional,
affected progenitors and differentiated neurons, and increased dramatically
with time (Fig. 1l and see Fig.
S1 in the supplementary material). Thus, we interpret our findings as
increased cell spread rather than a dorsal-to-ventral re-specification due to
the failure of Ptc1loop2 to
bind the HH ligand.
HH regulates the boundaries of the midbrain with adjacent tissues
In this study, we show that a consequence of HH blockade in the midbrain is
increased cell proliferation, resulting in a broadened MHB across which cell
mixing can occur (Kiecker and Lumsden,
2005; Vaage, 1969;
Zervas et al., 2005). Recent
evidence suggests that, rather than being a single boundary, the MHB may be a
compartment flanked by two boundaries, much like the zona limitans
intrathalamica (ZLI) in the diencephalon
(Kiecker and Lumsden, 2005).
The MHB is sharpened over time via the mutual repression of OTX2 and GBX2
(Zervas et al., 2005). Taken
together with our observations, these results support a role for HH signaling
in sharpening the MHB by inhibiting cell proliferation. Furthermore, although
controversial, the MHB is likely to be a lineage-restriction boundary, which,
like rhombomere boundaries, is somewhat `leaky' and permits a limited amount
of cell mixing (Fig. 5C,D)
(Jungbluth et al., 2001;
Zervas et al., 2005). The
increased cell mixing noted across the MHB following HH blockade in our
experiments therefore suggests a role for HH signaling in limiting such cell
mixing. This is corroborated in the Shh-/- mouse, in which
MHB cells can be found scattered several cell diameters away from the MHB
(J.L.F. and S.A., unpublished observations).
The requirement for HH in boundary maintenance is not confined to the MHB.
In Fig. 6, we noted that the DV
boundary and the accompanying serrate 1 expression are also perturbed as a
consequence of HH blockade and result in cell mixing. No patterning properties
are ascribed to the midbrain DV boundary yet, but Serrate and Notch-Delta
interactions have been implicated in DV patterning in the fly and vertebrate
limb and in the establishment of the apical ectodermal ridge, a signaling
center at the DV interface (Irvine and
Vogt, 1997). We conclude that maintaining the integrity and the
signaling properties of boundary regions, and therefore the territorial
integrity of the ventral midbrain, is an important function of HH
signaling.
Radial patterning and the cell autonomy of HH action within the ventral midbrain
In Fig. 3, we show that the
specification of the appropriate cell fates was not only blocked within
Ptc1loop2+ cells but also in
columns of Ptc1loop2 negative
cells that were radially aligned with them. In EGFP electroporations,
we show that cells within a single midbrain column can be cytoplasmically
continuous, raising the possibility of the transfer of small, undetectable
amounts of Ptc1loop2 between
these cells to block fate specification. In the cortex, lineally related cells
occupy similar radial columns and are cytoplasmically connected via gap
junctions (Chenn and McConnell,
1995; Noctor et al.,
2001). Intriguingly, gap junctions are also found among midbrain
progenitors (Fig. 3F). A recent
in vitro study has elegantly demonstrated the involvement of PTC1-mediated
induction of provitamin D3 in suppressing HH signaling in juxtaposed cells
(Bijlsma et al., 2006). This
model supports the extracellular transport of provitamin D3 in the
non-autonomous blockade of SMO in adjacent cells. However, provitamin D3 is a
small molecule (384.6 Da) and could pass through gap junctions from an
electroporated cell to its cytoplasmically connected neighbors to block
cell-fate specification. Thus, although the radial organization of the
midbrain may depend upon the alignment of clonally related cells, their
cytoplasmic connections may help explain why they share similar fates
following HH blockade.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2115/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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