Department of Biochemistry and Molecular Biophysics, Columbia University, 701 West 168th Street, New York, NY 10032, USA
e-mail: lz146{at}columbia.edu
Accepted 11 February 2005
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SUMMARY |
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Key words: Forebrain, Diencephalon, Signaling center, Shh, Chick
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Introduction |
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Along the dorsoventral axis, the assignment of neural cell fate is achieved
by a mechanism that is conserved along much of the length of the neural tube.
Signals provided by two polar cell groups, the floor plate ventrally and the
roof plate dorsally, impose distinct cell fates on intervening neural
progenitor cells (Lee and Jessell,
1999; Tanabe and Jessell,
1996
). Ventral cell fates are specified largely through a gradient
of Sonic hedgehog (Shh) activity that emanates from the floor plate
(Chiang et al., 1996
;
Ericson et al., 1995
;
Wijgerde et al., 2002
),
whereas the fate of many dorsal cell types is imposed by BMP family members
secreted from the roof plate (Liem et al.,
1997
; Nguyen et al.,
2000
; Timmer et al.,
2002
). This basic system of polarized Shh and BMP signaling from
midline cell groups operates within the spinal cord, hindbrain, midbrain and
caudal forebrain (reviewed by Briscoe and
Ericson, 2001
; Lee and
Jessell, 1999
). Moreover, even in the rostral forebrain, where
there is no overt floor plate or roof plate, Shh and BMP appear to impose
regional pattern on neural cells (Barth and
Wilson, 1995
; Dale et al.,
1997
; Ericson et al.,
1995
; Furuta et al.,
1997
; Golden et al.,
1999
; Hebert et al.,
2002
; Shimamura and
Rubenstein, 1997
).
In contrast to the uniformity of dorsoventral patterning, the specification
of rostrocaudal neural identity appears to obey a more fragmentary logic, with
distinct organizing centers operating over different rostrocaudal domains of
the neural tube. Three major signaling centers are known to control
rostrocaudal neural pattern: the anterior neural ridge (ANR), the isthmic
organizer (IsO) and the node. The ANR is positioned at the rostral extreme of
the neural tube and directs rostrocaudal cell fates in the telencephalon, in
part through the actions of Fgf8 (Houart
et al., 1998; Shimamura and
Rubenstein, 1997
). The IsO is positioned at the junction between
the midbrain and hindbrain (Broccoli et
al., 1999
; Li and Joyner,
2001
; Martinez-Barbera et al.,
2001
; Millet et al.,
1999
), and regulates the specification of cell types in the
midbrain and rostral hindbrain, through the secretion of Wnt1 and Fgf8
(reviewed by Liu and Joyner,
2001
). At more caudal levels of the neural tube that give rise to
the spinal cord, rostrocaudal positional identity is influenced by
node-derived signals, and Fgf8 is a major component of activity of the node
(Liu et al., 2001
). Thus, FGF
signaling is a common feature of the activity of three distinct rostrocaudal
organizing centers.
Within the diencephalon, however, the rostrocaudal patterning of cell types
occurs independently of signals provided by the ANR and the IsO
(Chi et al., 2003;
Jaszai et al., 2003
;
Shanmugalingam et al., 2000
),
and is likely to depend on signals provided by the ZLI, a prominent structure
that protrudes from the basal plate at the boundary between the prospective
ventral thalamus and the dorsal thalamus
(Kiecker and Lumsden, 2004
).
Shh is expressed within the ZLI (Echelard
et al., 1993
), and the acquisition of post-mitotic neural
identities in adjacent thalamic tissues emerges in the wake of the ventral to
dorsal progression of Shh expression within the ZLI
(Larsen et al., 2001
). Recent
studies demonstrated that Shh signals are required for the specification of
thalamic identitites, and that the likely source of these signals is the ZLI
(Hashimoto-Torii et al., 2003
;
Kiecker and Lumsden, 2004
).
Thus, the ZLI, as with the IsO and the ANR, is aligned perpendicular to the
main axis of the neural tube, but as with a dorsoventral organizing center,
the floor plate expresses Shh. In some respects then, the ZLI may be an
organizing center with properties characteristic of both rostrocaudal and
dorsoventral patterning centers.
Although the ZLI has been implicated in diencephalic patterning, it is as
yet unclear how the ZLI is formed. Some evidence about the early patterning
mechanisms that govern the initial position of ZLI formation has emerged. At
neural plate stages, cross-repressive interactions between the transcription
factors Six3 and Irx3 establish a boundary that anticipates the position of
formation of the ZLI (Kobayashi et al.,
2002). After neural tube closure, Wnt8b expression marks
the prospective ZLI and is flanked by domains of Lunatic fringe
(Lfng) expression (Garcia-Lopez
et al., 2004
). Furthermore, ectopic Lfng expression
represses ZLI formation, suggesting that the lateral limits of the ZLI are
normally constrained by these adjacent domains of Lfng expression
(Zeltser et al., 2001
).
Despite these insights, however, the factors responsible for inducing the ZLI,
in the context of these positional constraints, remain unknown.
In this study, I have examined the molecular mechanisms that control ZLI formation in an avian forebrain explant system. Through fate-mapping experiments, I show that the ZLI differentiates in the alar plate of the diencephalon in response to inductive signals derived from the basal plate. Shh signaling from the basal plate is required to initiate Shh expression within the ZLI, and, subsequently, long-range Shh signals provided by the basal plate and ZLI are needed for its dorsal progression. Finally, the dorsal limit of progression of the ZLI appears to be constrained in part by a limit in the range of Shh action, and in part by an opponent signal emanating from the dorsal diencephalon.
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Materials and methods |
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Whole-mount in situ hybridization and immunohistochemistry
Two-color in situ hybridization was performed as described
(Dietrich et al., 1997) with
the following probes: Shh, Ptc2
(Pearse et al., 2001
),
Lfng (Laufer et al.,
1997
), Wnt3a, Foxg1
(Bell et al., 2001
).
Immunohistochemistry was performed as described
(Yamada et al., 1993
) with
mouse anti-Shh (5E1) (Ericson et al.,
1996
) and sheep anti-GFP (Biogenesis). Alexa488- and
Cy3-conjugated secondary antibodies were obtained from Molecular Probes and
Jackson ImmunoResearch Laboratories, respectively. Images were obtained with a
Nikon E800 microscope.
Forebrain explant culture
Stage 13-14 chick heads were hemissected along the longitudinal axis of the
neural tube, the mesenchyme removed, and tissue containing the forebrain and
midbrain regions cultured in the presence of surrounding ectodermal tissues on
a Millipore filter in DMEM:F12 (Specialty Media) and 10% FCS (Hyclone). Before
fixation with 4% paraformaldehyde (PFA), the explants were attached to the
underlying filters with collagen (Cohesion).
Fate mapping and length measurements
DiI injection (Molecular Probes) and photooxidation were performed as
described (Psychoyos and Stern,
1996). Because the alar and basal plates could respond differently
to factors in the culture medium, where possible, I assessed the relative
length of the ZLI as a fraction of the length of the alar plate.
Hh inhibition and activation
To block Hh signaling, the monoclonal antibody 5E1 (Developmental Studies
Hybridoma Bank) was added to the culture medium at 25 ng/µl. Hh-Ag1.3 (10
nM to 1 µM; Curis) was added to the culture medium, which induced spinal
cord progenitors in a concentration-dependent manner
(Frank-Kamenetsky et al.,
2002).
Electroporation
Intact forebrain explants were electroporated in a Sylgard dish using a
concentric bipolar electrode (FHC, Bowdoinham, ME) to produce focal regions of
misexpression. A 2.5 mg/ml DNA solution was injected above the target tissue
and four 50-msec pulses of 15V were delivered by a T820 electrosquareporator
(BTX, San Diego, CA). mShh-CD4 (Yang et
al., 1997) and full-length mShh
(Riddle et al., 1993
) were
cloned into the pCAGGS vector (Fukuchi et
al., 1994
). Each plasmid solution contained a mixture of 0.25
mg/ml pCAGGS-GFP plasmid (Momose et al.,
1999
). Explant manipulations were performed after electroporation.
Explants were incubated at 37°C for 48 hours, embedded in collagen, and
fixed as described above. Electroporated cells were visualized by staining
with a sheep-anti-GFP antibody (Biogenesis).
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Results |
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To examine the source and identity of signals that initiate the program of
ZLI differentiation, I analyzed diencephalic development in an in vitro
culture system (described in Materials and methods). After 48 hours, these
explants achieved a size equivalent to that of stage 20 embryos grown in ovo.
The onset of ZLI differentiation in vitro, as marked by Shh and
Ptc2 expression, extended dorsally at 13 µm per hour
(Fig. 1D-I,K), reaching a final
position in the alar plate similar to that observed in stage 20 embryos in
ovo. Thus, the progression of ZLI differentiation in vitro appears to proceed
in a similar manner to that in vivo, albeit at a slightly slower rate.
Induction of Shh expression in the ZLI by signals from the basal plate
I next investigated whether the progressive dorsal expansion of
Shh expression in the ZLI reflects the migration of
Shh-expressing cells from the basal plate, or the induction of ZLI
differentiation in cells already positioned within the alar plate. To
determine whether cells from the basal plate contribute to the ZLI, I injected
carbocyanine dye (DiI) focally into the basal plate of stage 13-14 explants.
The precise location of labeled cells with respect to the ZLI was determined
by photo-conversion of DiI and analysis of the position of cells containing
DiI-induced precipitate in relation to the domain of Ptc2 expression
(Fig. 2A). After DiI injections
into the basal plate, few if any labeled cells were detected in the ZLI after
48 hours (n=24; Fig.
2C-E). Thus, the dorsal migration of Shh-expressing
basal-plate cells does not appear to underlie the progressive dorsal expansion
of Shh expression that is predictive of ZLI differentiation.
|
To address the origin of signals involved in ZLI differentiation, I assessed whether the basal plate is required for the induction of Shh expression in the ZLI. Using the sulcus at the alar/basal plate boundary as a guide, I excised the ventral diencephalon and assessed ZLI formation in resulting dorsal explants grown for 24-48 hours. Two sets of dorsal (D) explants were analyzed: D2/3 explants, which contained most of the alar plate, and D1/2 explants, which lacked the ventral-most region alar plate (Fig. 3A). To confirm the accuracy of dissection, I assessed Shh and Ptc2 expression in dorsal explants fixed immediately after dissection (t=0 hours). In addition, to determine the ventral boundary of dorsal explants relative to the basal plate, I assessed Ptc2 expression in the complementary ventral region of excised diencephalic tissue (V explants) at t=0. All V1/2 and V1/3 explants expressed Shh and Ptc2 at the time of dissection, but expression of these genes was never detected in D1/2 and D2/3 explants (n=19, 20 respectively), providing evidence that dorsal explants do not contain basal-plate tissue (Fig. 3B,C,F,G,J,K).
|
I therefore considered whether the ZLI-inducing activity of the ventral alar plate is produced locally, or whether it is the result of signals secreted from the basal plate. To assess whether the basal plate is a source of Shh-inducing signals, I monitored Shh expression in D1/2 explants co-cultured with basal-plate tissue (Fig. 4A). D1/2 explants never expressed Shh when grown alone, but they did express Shh when the basal plate was grafted to the ventral side of the D1/2 explant (n=8/9; Fig. 4B-C). This finding supports the idea that the basal plate is the source of a signal(s) that initiates Shh expression in adjacent alar-plate tissue. In turn, the emergence of Shh expression in a ZLI-like domain in D2/3 explants grown alone suggests that the dorsal progression of ZLI differentiation rapidly acquires independence from ongoing signaling from the basal plate.
|
I also assessed the effect of blocking Hh signaling in intact forebrain explants that were not subdivided (intact explants). Exposure of stage 13-14 forebrain explants to MAb-5E1 for 48 hours did not alter Shh expression in the basal plate, supporting the view that once Shh expression in the basal plate has been established, its persistence does not require ongoing Hh signaling. By contrast, the domain of Shh expression within the ZLI was dramatically reduced in all intact forebrain explants grown in the presence of MAb-5E1 (mean reduction of 70-80%; n=52; Fig. 5B,C). The persistence of Shh expression in the ventral domain of the ZLI of stage 13-14 explants exposed to MAb-5E1 could reflect the prior influence of Shh signaling. To test this, forebrain explants were exposed to MAb-5E1 at stages 10-12, and under these conditions Shh expression in the ZLI domain was virtually eliminated (n=9/9; Fig. 5D). Together, these findings support the idea that Shh signaling is required for the initiation of a program of ZLI differentiation.
|
Spatial constraints on ZLI induction by Shh
Shh expression in the basal plate extends along the length of the
forebrain, whereas expression in the alar plate is restricted to the narrow
stripe predictive of the ZLI. The competence of alar-plate cells to express
Shh may therefore be restricted to the presumptive ZLI. To assess
this, I examined the consequences of widespread exposure of diencephalic
alar-plate cells to Hh activity. D1/2 explants were cultured in the presence
of the small molecule Hh agonist 1.3 (Hh-Ag1.3) for 48 hours
(Frank-Kamenetsky et al.,
2002). Shh was expressed selectively in a stripe of cells
that corresponded to the position of the ZLI
(Fig. 6D). Thus, alar-plate
cells rostral and caudal to the prospective ZLI are not competent to express
ZLI markers upon exposure to Hh agonists. This finding implies the existence
of a mechanism that confines Shh expression to the ZLI.
|
A signal from the dorsal diencephalon inhibits Shh induction of ZLI differentiation
To assess the ability of diencephalic tissue to form a ZLI in the absence
of potentially inhibitory signals from the dorsal diencephalon, I generated
intermediate (I) explants by removing the dorsal fifth of the neural tube from
dorsal (D) explants (Fig. 7A).
To facilitate survival of the narrow intermediate explants, the ventral
boundaries of the dorsal and intermediate explants were shifted to a position
between D1/2 and D2/3 explants. Wnt3a is expressed in the dorsal
midline of the diencephalon and midbrain and in the prospective thalamus from
stage 12-13 (data not shown), and this marker was therefore used to assess the
accuracy of the dissection and the maintenance of a diencephalic identity in
the explant. The dorsal domain of Wnt3a expression was observed in
both intact and dorsal explants, but was completely lost from intermediate
explants (Fig. 7E), providing
evidence that the dorsal extreme of the neural tube was removed completely.
Moreover, Wnt3a expression was detected in intact, dorsal and
intermediate explants (Fig.
7C-E) after two days in culture, suggesting that a diencephalic
identity was maintained in the absence of ongoing signaling from ventral
and/or dorsal tissues.
|
I also examined the ventral to dorsal propagation of ZLI differentiation in
dorsal versus intermediate explants elicited by a localized source of Hh
signals, a scenario that is likely to approximate the process of ZLI
differentiation in vivo. Stage 13-14 forebrain explants were focally
electroporated with a membrane-tethered form of mShh (mShhCD4)
(Yang et al., 1997). Dorsal
explants electroporated with mShhCD4 did not express Shh at any
position in the explant (n=43;
Fig. 7F). Because mShhCD4 is
active in other assays (data not shown), this finding suggests that
electroporation of mShhCD4 achieves a low level of Shh signaling. By contrast,
in mShhCD4-expressing intermediate explants, Shh was expressed throughout the
ZLI domain, even when electroporation was restricted to the ventral aspect of
the explant (n=4; Fig.
7G). Similar results were obtained with electroporation of a
secreted form of mShh in intermediate versus intact forebrain explants (data
not shown). These data provide additional support for the idea that the dorsal
diencephalic region is the source of a factor that can block the progression
of ZLI differentiation.
To examine more directly whether a dorsally derived signal opposes ZLI
formation, I assessed whether grafts of dorsal diencephalic tissue can
suppress the propagation of ZLI differentiation. The dorsal fifth of the
diencephalon was cultured adjacent to the prospective thalamic territory in
stage 13-14 explants, and the location of the grafted tissue relative to the
ZLI after two days in culture was determined by in situ hybridization with
probes against Wnt3a and Shh, respectively
(Fig. 8A). Dorsal diencephalic
tissue inhibited the propagation of Shh expression in the adjacent
ZLI beyond the position of the graft (n=16;
Fig. 8C,E). Control grafts of
intermediate telencephalic tissue, marked by Foxg1 expression
(Bell et al., 2001), did not
affect the expression of Shh in the ZLI (n=9;
Fig. 8B,D,F). Taken together,
the complementary effects of the removal or addition of dorsal diencephalic
tissue provide strong evidence that it provides a signal that opposes the
propagation of ZLI differentiation.
|
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Discussion |
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Shh signaling and the progression of ZLI differentiation
Previous in ovo cell-labeling studies have revealed that the ZLI forms
within a narrow lineage-restricted compartment that is demarcated by borders
of Lfng expression (Zeltser et
al., 2001). However, in these in ovo studies the initial position
of labeled cells was not determined precisely, and thus the cellular origin of
the ZLI remained unclear. My findings help to resolve the issue of whether the
ZLI emerges through the directed dorsal migration of Shh-expressing
basal-plate cells or from de novo induction of ZLI character within alar-plate
cells. The present fate-mapping studies in forebrain explants provide strong
evidence that the program of ZLI differentiation is initiated in cells that
occupy the ventral region of the alar plate, and subsequently propagates
dorsally in the absence of extensive cell migration. These findings are
consistent with a recent fate-mapping study that locates prospective ZLI
territory in the alar plate of stage 9-10 chick embryos
(Garcia-Lopez et al.,
2004
).
In principle, the inductive signals that initiate ZLI differentiation could
be transferred in planar fashion from adjacent diencephalic tissues, a
suggestion that has been favored
(Echevarria et al., 2003;
Echevarria et al., 2001
;
Kobayashi et al., 2002
), or
alternatively could be transmitted vertically from the basal plate. The
observation that cells in the ventral-most region of the alar plate, present
in D2/3 explants but not in D1/2 explants, are required to sustain a program
of ZLI differentiation provides evidence that ventrally derived signals are
involved. A plausible candidate for a ventral ZLI-initiating signal derived
from the basal plate is Shh. The ability of basal-plate tissue to induce ZLI
differentiation in adjacent dorsal explants was severely reduced by a blockade
of Shh signaling within the basal plate, suggesting that Shh signaling from
the basal plate is required to initiate ZLI differentiation.
In contrast to the absolute requirement for Shh signaling in ZLI formation
in the chick, studies in the zebrafish system provide evidence that a
subpopulation of cells in the ZLI is independent of Hh or basal plate-derived
signals. Whereas treatment of chick forebrain explants with the Hh pathway
inhibitors MAb 5E1 or cyclopamine (data not shown) abolishes Shh
expression in the ZLI, zebrafish embryos grown in the presence of cyclopamine
exhibited a reduced domain of Shh expression in the ZLI
(Mathieu et al., 2002).
Divergent conclusions have emerged from analyses of ZLI formation in zebrafish
smoothened (smu) mutants, in which signaling downstream of
all Hh family ligands is disrupted
(Holzschuh et al., 2003
;
Mathieu et al., 2002
).
Holzschuh et al. observed a requirement for Hh signaling at 24 hpf that was
not observed at 28 hpf by Mathieu et al. This discrepancy could reflect a
delay in ZLI formation in the smu mutants, and/or bona fide
differences in the smu alleles analyzed. The presence of a reduced
domain of Shh expression at the position of the ZLI in smu
mutants at 28 hpf could be explained by a gradual loss of the activity of
maternal Smu protein, which coincides with the period of ZLI formation
(Varga et al., 2001
). This
phenotype would be analogous to the reduced ZLI I observed when intact
explants were grown in the presence of Shh-blocking antibodies
(Fig. 5C,D). Alternatively, the
presence of an Hh-independent subpopulation of cells in the ZLI in zebrafish
could reflect a difference between the two species. A small domain of
Shh expression at the position of the ZLI is also observed in 26% of
zebrafish cyclops mutants, which lack a basal plate in the
diencephalon (Sampath et al.,
1998
). Shh expression in these cells could result from
inefficient Shh signaling from the subjacent notochord, or could represent a
subpopulation of the ZLI that is independent of basal plate-derived signals.
Although the nature of the ZLI phenotype in zebrafish mutants needs to be
analyzed further, the existence of an Hh-independent subpopulation in the ZLI
could reflect species differences analogous to those observed in another
Shh-expressing tissue, the floor plate (reviewed by
Strahle et al., 2004
). In the
chick and mouse, specification of both the medial floor plate (MFP) and
lateral floor plate (LFP) are Hh-dependent
(Chiang et al., 1996
;
Ericson et al., 1996
). By
contrast, LFP formation in zebrafish is dependent on Hh signaling, whereas
formation of the MFP is not (Chen et al.,
2001
; Karlstrom et al.,
1999
; Schauerte et al.,
1998
; Varga et al.,
2001
).
I have found that ZLI differentiation in the alar plate progresses in the absence of sustained signaling from the basal plate, raising the issue of how ZLI differentiation is propagated dorsally. The dorsal propagation of ZLI differentiation can be arrested by a blockade of Hh signaling late in the period of explant culture, suggesting a requirement for ongoing Shh signaling during the period of ZLI differentiation. This finding does not support the possibility that Hh merely initiates ZLI differentiation, with its dorsal progression depending on a distinct secondary signal. Since there is not a continuous requirement for a basal-plate source of Shh for propagation of ZLI differentiation within alar-plate explants, the ZLI is the most likely source of Shh involved in this later phase of diffusible signaling. Taken together, my findings support the idea that basal plate-derived Shh signals initiate ZLI differentiation in the alar plate, and that Shh signals from the ZLI itself participate in the subsequent dorsal progression of ZLI differentiation.
Inhibitors restrict Shh expression to the ZLI
Shh is expressed throughout the rostrocaudal axis of the diencephalic basal
plate, raising the issue of how the rostrocaudal position of the ZLI is
determined, with respect to the much broader ventral domain of expression of
Shh, its inductive signal. At neural plate stages, several genes are
expressed in domains that have boundaries at the position of the future ZLI,
and some of these genes are likely to have a role in restricting ZLI
differentiation. The domains of Six3 and Irx3 expression in
the neural plate abut each other, and could influence the position of ZLI
formation by establishing zones of non-competence in flanking neural tissue
(Kobayashi et al., 2002).
At later developmental stages at which overt ZLI differentiation occurs,
Six3 expression regresses rostrally so that it no longer abuts the
Irx3 domain, consistent with the idea that other factors directly
regulate the initiation and progression of ZLI differentiation. At stage 13,
Lfng and Wnt3b are expressed in complementary domains that
demarcate the borders of the ZLI
(Garcia-Lopez et al., 2004;
Garda et al., 2002
;
Zeltser et al., 2001
).
Moreover, ectopic Lfng disrupts cell sorting at the compartment boundaries and
prevents Shh expression in the ZLI
(Zeltser et al., 2001
),
suggesting that Lfng constrains ZLI differentiation in flanking thalamic
tissues by repressing the potential for Shh expression. The
maintenance of normal borders of Lfng expression in the absence of
Shh signals is consistent with the idea that a distinct set of developmental
cues, possibly provided by Six3, Irx3 and Wnts, establishes a prepattern in
the diencephalon (Braun et al.,
2003
; Kobayashi et al.,
2002
; Kiecker and Lumsden,
2004
).
The ZLI never encroaches into the dorsal extreme of the diencephalon, and
dorsal diencephalic tissue does not respond to Hh exposure with ZLI induction,
suggesting that dorsal diencephalic tissue is normally unable to initiate ZLI
differentiation. Following the removal of the dorsal extreme of the neural
tube, the enhanced sensitivity of dorsal regions of the diencephalon to Shh
signaling suggests that this tissue is exposed to a signal that suppresses ZLI
differentiation. Moreover, the ventral-to-dorsal gradient of sensitivity of
diencephalic tissue to different levels of Hh signaling during ZLI induction
suggests that this inhibitor acts in a graded manner. The suppression of ZLI
propagation adjacent to grafts of dorsal diencephalic tissue provides
additional evidence for a secreted inhibitory factor. Candidate factors for
such a secreted dorsal inhibitor include members of the BMP/GDF
(Furuta et al., 1997;
Golden et al., 1999
;
Lee and Jessell, 1999
) and Wnt
(Garda et al., 2002
;
Hollyday et al., 1995
)
families of signaling molecules. In caudal neural tissue, long-range BMP
signals from the surface ectoderm and prospective roof plate have been
proposed to antagonize floor-plate induction by Shh secreted from the
notochord (Patten and Placzek,
2002
). Simultaneously, BMPs secreted from the roof plate influence
late patterning in the brain and spinal cord by antagonizing Shh signaling
(Liem et al., 2000
;
Liem et al., 1995
;
Ohkubo et al., 2002
).
The ZLI as a hybrid organizer
My findings suggest that the ZLI possesses features of both rostrocaudal
and dorsoventral signaling centers. The node, IsO, ANR and ZLI secrete
signaling molecules, notably FGFs, which control cell identities along the
rostrocaudal axis. The ZLI, as with the IsO, is oriented perpendicular to the
main axis of the neural tube, yet is distinct from other rostrocaudal
organizers in that it expresses Shh, a signaling molecule associated with the
two main dorsoventral organizers, the notochord and floor plate.
Although the ZLI influences the acquisition of thalamic cell identities
along the rostrocaudal axis, the mechanism regulating ZLI differentiation
resembles that involved in establishing sequential dorsoventral organizers. In
particular, ZLI differentiation is induced in the diencephalic alar plate
through a mechanism reminiscent of the induction of lateral floor-plate cells
(LFP) in the ventral-most region of the neural tube. Here, Shh signaling from
the MFP is required to induce LFP differentiation in adjacent cell populations
in the neuroepithelium (Charrier et al.,
2002; Ding et al.,
1998
; Matise et al.,
1998
; Schauerte et al.,
1998
). Moreover, floor-plate signaling appears to be opposed by
long-range signals from the dorsal neural tube
(Patten and Placzek, 2002
). In
contrast to the LFP, which occupies only a few cell layers in the ventral
neural tube, long-range Shh signaling leads to the extensive propagation of
ZLI differentiation along the dorsoventral axis of the diencephalon. Cells in
the MFP and LFP are likely to play redundant roles in ventral patterning in
the neural tube (Odenthal et al.,
2000
). By contrast, Shh signals emanating from the basal plate and
ZLI appear to have distinct functions in patterning diencephalic cells along
orthogonal axes. These dual and orthogonal sources of Shh signaling in the
diencephalon may contribute to the emergence of the complex nuclear
organization of the thalamus.
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ACKNOWLEDGMENTS |
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