Department of Human Genetics, University of Chicago, 920 East 58th Street, CLSC 319, Chicago, IL 60637, USA
* Author for correspondence (e-mail: kmillen{at}genetics.bsd.uchicago.edu)
Accepted 11 February 2004
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SUMMARY |
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Key words: Lmx1a, dreher, Roof plate, Bmp, Neural crest, Developing spinal cord, Chick, Mouse
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Introduction |
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The search for molecular signals involved in dorsal neural cell type
specification has identified several secreted candidate molecules, including
Bmp4/7 and Wnt1 (Liem et al.,
1997; Garcia-Castro et al.,
2002
); however, their precise role in roof plate specification is
poorly understood. Even less is known about intrinsic factors controlling the
specification of roof plate cells. Only a few transcription factors, including
Pax3, Pax7 and Msx1/2, have been implicated in roof plate development.
Importantly, however, these molecules are also involved in the development of
neural crest and dorsal interneurons (Lee
and Jessell, 1999
; Knecht and
Bronner-Fraser, 2002
). This suggests that other, roof
plate-specific, intrinsic factors must separate the roof plate fate from other
dorsal cell fates.
Recently we analyzed the spontaneous neurological mouse mutant
dreher, which fails to develop roof plate, and identified
inactivation of the LIM-homeodomain transcription factor Lmx1a as the
cause of this phenotype (Millonig et al.,
2000). This analysis demonstrated that Lmx1a is essential for
either the induction or maintenance of roof plate cells. In this paper we
demonstrate that Lmx1a is sufficient for withdrawing dorsal spinal cord
progenitors from the cell cycle and simultaneously driving them to
differentiate into functional roof plate cells in the dorsal neural tube.
Interestingly, our data demonstrate that Lmx1a is not involved in suppression
of neural crest cell fate. At the same time, Lmx1a represses Math1 and
prevents generation of dI1 dorsal interneurons in a cell-autonomous fashion,
thus distinguishing the roof plate and dI1 interneuronal programs in the
dorsal neural tube. Finally, our results indicate that Bmps are necessary and
sufficient for inducing Lmx1a and roof plate cells in the neural tube, and
that Lmx1a is a major mediator of early Bmp signaling in developing spinal
cord.
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Materials and methods |
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Expression constructs
cDNAs encoding full-length mouse wild-type and dreherJ
point mutation alleles of Lmx1a
(Millonig et al., 2000),
Bmp4 and Bmp7 were cloned upstream of IRES-EGFP in the pCIG
expression vector (Megason and McMahon,
2002
). Bmp4 was also expressed using pMiwII-Bmp4
(Kishimoto et al., 2002
). No
differences between pCIG-Bmp4 and pMiw-Bmp4 were detected. Activated human
Bmp receptor Ia, chick Noggin, human dnTCF4 and
mouse dnWnt1 were expressed from pCAGGS-caBmpr-Ia
(Timmer et al., 2002
), a
Noggin expression vector (L. Niswander, unpublished, a gift from L.
Niswander), pCIG-dnTcf4 (Megason and
McMahon, 2002
) and pC1-neo-dnWn1
(Garcia-Castro et al., 2002
).
In all experiments an appropriate empty vector was used as a control.
In-ovo electroporation
Supercoiled plasmid DNA was injected into the lumen of the rostral neural
tube of stage 10 embryos at a concentration of 1-2 mg/ml in water together
with 50 µg/ml Fast Green dye (if the injected plasmid did not express EGFP,
empty pCIG at a concentration of 0.2 mg/ml was added to the injected
solution). Electroporation was performed as previously described
(Megason and McMahon, 2002).
The eggs were sealed and allowed to develop for a further 15-48 hours. Embryos
with high levels of GFP fluorescence in the neural tube were processed for
further analysis.
Neural explants
Intermediate neural plate explants from electroporated and control neural
plates were isolated (Liem et al.,
1995; Liu and Jessell
1998
) and cultured in serum-free medium as described by
Garcia-Castro et al. (2002
).
Explants of mouse neural tubes were dissected in dispase and cultured as
described by Lee et al.
(1998
). Induction experiments
were performed in the presence of 100 ng/ml human Bmp4 (R&D Systems).
Immunohistochemistry and in-situ hybridization
Immunohistochemistry was performed on frozen sections and in whole-mount as
previously described (Helms and Johnson,
1998; Sela-Donenfeld and
Kalcheim, 1999
). Rabbit anti-Lmx1a antibody (M. German,
unpublished) was a gift from M. German. Rabbit anti-LH2A/B
(Lee et al., 1998
) and rabbit
anti-Maf B antibodies (Pouponnot et al.,
1995
) were provided by T. Jessell. The rabbit antibodies
anti-Math1 (Helms and Johnson
1998
), anti-Sox9 (Stolt et
al., 2003
) and anti-Lbx1
(Muller et al., 2002
) were
provided by J. Johnson, M. Wegner, and T. Muller and C. Birchmeier,
respectively. Mouse anti-HNK-1 antibody was obtained from ATCC. Mouse
anti-MitF and rabbit anti-GFP antibodies were purchased from Neomarkers and
Biocompare, respectively. The following primary mouse antibodies were obtained
from the Developmental Studies Hybridoma Bank (The University of Iowa,
Department of Biological Sciences, Iowa City, IA 52242): anti-Islet1 (51.4H9),
anti-Lim1/2 (4F2), anti-Slug (62.1E6), anti-Ap2 (3B5), anti-RhoB (54.4H7),
anti-Msx1/2 (4G1), anti-Pax7, anti-Pax6, anti-Math1 and anti-neurofilament
(2H3). Secondary species appropriate antibodies with Texas Red, FITC or HRP
conjugates were obtained from Jackson Immunological.
In-situ hybridization was performed on sections and in whole-mount as
previously described (Kos et al.,
2001; Helms and Johnson
1998
) using digoxigenin-labeled riboprobes to chick Gdf7,
Bmp4, Wnt1 and rat Isl1 provided by T. Jessell (Gdf7
and Isl1), P. Brickell (Bmp4) and A. McMahon
(Wnt1). Sections were digitally photographed with an AxioCam on a
Zeiss AxioPlan 2 microscope and processed using Adobe Photoshop.
BrdU labeling
BrdU labeling was performed as previously described
(Megason and McMahon 2002;
Dickinson et al., 1994
). For
double labeling, frozen sections were first processed for immunostaining, then
refixed and processed for BrdU staining with mouse anti-BrdU antibody (Zymed).
Some sections were also counterstained with propidium iodine to visualize the
tissue.
Measurements and statistical analyses
All sections analyzed were from the region between the forelimbs and hind
limbs, except the section shown in Fig.
1A, which were taken at the hind limb level. All results described
here were replicated in at least three independent embryos or explants.
Analysis of the effect of ectopic expression of Lmx1a on the cellular
distribution along the medio-lateral axis of the chick neural tubes was
conducted as illustrated in Fig.
4C,D. The number of BrdU/Lmx1a double positive cells was counted
using ten serial 12 µm transverse sections of the spinal cord of wild-type
and dreher embryos. The Lmx1a positive area was calculated using
these same BrdU/Lmx1a labeled sections by the method described by Dickinson et
al. (1994). All quantitative
data are expressed as the mean±s.e.m. Statistical significance was
determined by two-tailed t-test. * indicates P<0.01.
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Results |
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Morphological analysis of cross sections of chick neural tubes expressing
exogenous Lmx1a 48 hours after electroporation revealed expansion of the
dorsal midline domain in 20/25 embryos
(Fig. 2B,C and see Fig. S1 at
http://dev.biologists.org/supplemental/).
This phenotype has never been observed in embryos electroporated with EGFP
alone (n=20) or unelectroporated embryos (n=15)
(Fig. 2A,D,E). Roof plate
marker analysis (Liem et al.,
1997; Lee et al.,
1998
; Hollyday et al.,
1995
) revealed that there was an approximately fourfold increase
in the number of MafB-positive dorsal cells in embryos electroporated with
Lmx1a-IRES-EGFP (n=10 embryos) compared with embryos electroporated
with EGFP alone (n=8 embryos)
(Fig. 2B-F). Expression domains
of Gdf7, Bmp4 and Wnt1 were also ectopically expanded on the
Lmx1a electroporated side (13/15 embryos)
(Fig. 2L-Q). Moreover, we found
that markers of Bmp activity, the Msx1/2 proteins
(Timmer et al., 2002
), were
shifted ventrally (13/15 embryos) (Fig.
2G-K) further indicating an expanded domain of Bmp signaling
activated by exogenous Lmx1a in the dorsal neural tube. The ectopic expression
of roof plate markers was limited to the most dorsal region of the neural
tube. No expression of roof plate markers was detected in intermediate and
ventral areas of the neural tubes, in spite of the high levels of exogenous
Lmx1a expression (n=25 embryos)
(Fig. 2C,I and data not shown).
To demonstrate that Lmx1a was not simply expanding already specified
roof plate, we conducted explant experiments. We determined that Lmx1a could
induce roof plate cells in naive neural tissue in vitro, as defined by
MafB/Pax7 double staining (Liem et al.,
1997
), when overexpressed in chick stage 10 intermediate neural
plate explants (n=8 explants)
(Fig. 2R-V). Analysis of dorsal
neural tube or explants electroporated with Lmx1a-IRES-EGFP revealed extensive
overlap of the GFP signal and the expression of the roof plate markers
investigated (Fig. 2C,U and
data not shown), suggesting that Lmx1a induces roof plate
cell-autonomously.
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Analysis of BrdU incorporation at 18 hours after electroporation showed
that many cells expressing exogenous Lmx1a were BrdU positive and
were still randomly distributed along the medio-lateral axis of the neural
tube (Fig. 4E,F). At 36 hours
after electroporation, however, there was a marked reduction in the number of
BrdU-incorporating cells in the Lmx1a electroporated regions compared
with the control side of the neural tube (n=9 embryos). At this later
time, the vast majority of cells expressing exogenous Lmx1a were
BrdU-negative, including cells located in the neural tube and migrating neural
crest cells (Fig. 4G,H). Cells
electroporated with EGFP alone were still proliferating (n=8 embryos)
(data not shown). TUNEL labeling to determine the number of cells undergoing
apoptosis showed no differences between Lmx1a electroporated and
control sides of the neural tube either at 18 hours after electroporation or
36 hours after electroporation (n=7 and 5 embryos, respectively, data
not shown). Taken together, our data indicate that, when overexpressed in
chick neural tube, Lmx1a withdraws neural progenitors from the cell cycle.
This effect is not immediate but is delayed by at least 18 hours.
Interestingly, expression of exogenous Lmx1a does not change the
identity of intermediate and ventral neural cells, since they continue to
express appropriate subtype-specific markers, including the dI4-6 interneuron
marker Lbx1 (Muller et al.,
2002; Gross et al.,
2002
) and the motoneuron marker Isl1
(Ericson et al., 1992
) (data
not shown).
To further investigate the role of Lmx1a in the cell cycle regulation of
roof plate progenitors we returned to the dreher mouse model. Using
Lmx1a-specific antibodies we could detect mutant Lmx1a protein in dorsal
spinal cord of E9-10 drJ/drJ embryos
(Fig. 4L). This observation
allowed us to use BrdU/Lmx1a double labeling to compare number of
proliferating cells on the dorsal midline of the neural tube in
drJ/drJ and wild-type embryos. Our analysis
revealed that at E9.25 dorsal Lmx1a-positive cells were proliferative, and no
difference between drJ/drJ and wild-type
embryos was detected at this stage (data not shown). At E10.0, however, the
number of BrdU/Lmx1a-double-positive cells in spinal cord of
drJ/drJ embryos (n=5 embryos) was
threefold higher than that of wild-type littermates (n=8 embryos)
(Fig. 4I-M). In addition, the
mean area occupied by Lmx1a positive cells in
drJ/drJ embryos at E10.0 was approximately 2.2
times larger then that observed in wild type littermates
(Fig. 4J,L,N). This increase in
Lmx1a-positive area correlates well with the increase in number of
BrdU-positive cells in this region found in
drJ/drJ embryos. Since no change in the rate of
apoptosis has been detected in dorsal spinal cord of
drJ/drJ embryos
(Millen et al., 2004), this
indicates that an increase in proliferation of Lmx1a-positive cells accounts
for the increase in the area of Lmx1a-positive territory in
drJ/drJ embryos. Thus, our gain-of-function
experiments in chick and loss-of-function studies in mouse indicate that Lmx1a
is necessary and sufficient for negatively regulating proliferation of dorsal
neural progenitors in vivo.
Lmx1a is not critically involved in the early neural crest specification program
Since roof plate cells and neural crest cells derive from a common
progenitor in the neural folds and the early dorsal neural tube
(Bronner-Fraser and Fraser,
1988; Echelard et al.,
1994
), we investigated the role of Lmx1a in neural crest
formation. Analysis of the chick embryos electroporated with Lmx1a 18
hours after electroporation showed that ectopic expression of this gene failed
to significantly change numbers of early neural crest cells, as indicated by
several markers staining including Slug, Sox9 and Ap2 (n=8-10 embryos
for each marker) (Fig. 5A-C).
Also, there was no change in the expression of migratory neural crest markers
RhoB and HNK1 that were investigated 48 hours after electroporation
(n=9 embryos) (Fig.
5D-G and data not shown). In addition, Lmx1a did not
induce expression of any of these neural crest markers when ectopically
expressed in intermediate neural plate explants in vitro (n=7
explants, data not shown). Finally, chick embryos electroporated with
Lmx1a-IRES-EGFP revealed many GFP-positive cells migrating away from the
neural tube (n=9 embryos) (Fig.
5E,G), suggesting that Lmx1a is insufficient for preventing neural
crest migration.
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Lmx1a distinguishes roof plate and dI1 interneuron programs in the dorsal spinal cord
Next we investigated the cell-autonomous effects of exogenous
Lmx1a on development of dI1, dI2 and dI3 interneurons in chick dorsal
spinal cord. Since we have demonstrated that roof plate induced by exogenous
Lmx1a in the most dorsal regions of the neural tube non-autonomously
affects adjacent dorsal interneuronal specification
(Fig. 3), we limited our
analysis to neural tubes expressing exogenous Lmx1a only in regions
normally populated by dorsal interneurons
(Fig. 6D,F). This revealed that
many cells expressing exogenous Lmx1a still express Lim1/2 or Islet1
(n=4-6 embryos for each marker)
(Fig. 6C-F), indicating that
exogenous Lmx1a cannot cell-autonomously alter dI2 and dI3
interneuronal fates. By contrast, we very rarely observed cells co-expressing
LH 2A/B and exogenous Lmx1a (n=10 embryos)
(Fig. 6A,B), although many
cells co-expressed LH2 A/B and EGFP when a GFP control was used (n=5
embryos) (data not shown). This indicates that Lmx1a can cell-autonomously
suppress the dI1 interneuronal fate when overexpressed in the chick developing
spinal cord. To gain insight into the mechanism of this suppression, dI1
interneuron progenitors characterized by expression of Cath1 (chicken homolog
of Math1) (Helms and Johnson,
1998; Lee et al.,
1998
) were examined in electroporated samples (n=5
embryos). Again, no overlap between exogenous Lmx1a and Cath1 was
detected (Fig. 6G,H). Thus, the
cell-autonomous inhibition of dI1 interneuronal fate by Lmx1a has already
occurred at the progenitor stage.
To further test the cell-autonomous inhibition of dI1 interneuron progenitors by Lmx1a, we investigated expression of Math1 in mice. In wild-type E10.0 mouse spinal cord, Math1-positive dI1 interneuron progenitors are located directly adjacent to the ventral boundary of the roof plate and there is no overlap between the Lmx1a and Math1 expression domains at cellular level (Fig. 6I,J). In dorsal spinal cord of drJ/drJ E10.0 embryos, however, Math1 positive dI1 dorsal interneuronal progenitors were located in the region normally occupied by the roof plate, and many of them were also Lmx1a-positive (Fig. 6K,L). Taken together with our chick overexpression studies, these data indicate that Lmx1a cell-autonomously represses Math1, preventing roof plate cells from adopting the dI1 interneuronal progenitor cell fate.
The role of Bmp and Wnt signaling in activation of Lmx1a expression and roof-plate development in vivo
We next addressed which signal might mediate activation of Lmx1a
expression and roof plate formation in the developing chick spinal cord. Our
studies were focused on Bmp and Wnt proteins since they are expressed in
epidermal ectoderm at the time of roof plate formation and can induce
specification of several dorsal cell types in neural plate explants when added
to culture medium (Liem et al.,
1997; Muroyama et al.,
2002
; Garcia-Castro et al.,
2002
). First, we expressed Noggin, a Bmp4-secreted
inhibitor (Liem et al., 1997
),
to downregulate endogenous Bmp signaling in chick stage 10 neural plate.
Marked inhibition of Lmx1a expression, as well as inhibition of other roof
plate markers, including MafB and Bmp4, was detected 18 hours after
electroporation with Noggin-expressing plasmid (5/7 embryos)
(Fig. 7A-E).
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Induction of Lmx1a and roof plate by Bmp signaling
To investigate if Bmp signaling is sufficient for inducing Lmx1a
expression and ectopic roof plate formation, we expressed mouse Bmp4,
Bmp7 or an activated Bmp receptor, BmprIa, in chick developing
spinal cords using the same parameters and conditions as described for
Lmx1a. Ectopic expression of either construct resulted in a similar
phenotype, including significant hypocellularity and increased apoptosis (as
detected by TUNEL) within the electroporated regions (8/8 embryos). At the
same time, induction of Lmx1a along the whole dorsoventral axis of the neural
tube, excluding only the most ventral regions, was observed
(Fig. 8A,B and data not shown).
Surprisingly, activation of Bmp signaling induced expression of both MafB and
Lmx1a to the same broad extent along the dorsoventral axis of the neural tube
(compare adjacent sections shown on Fig.
8A,B and Fig. 8C,D). This was in contrast to exogenous Lmx1a,
which could induce MafB only in a restricted dorsal domain of the neural tube
(n=8 embryos). These data suggest that Bmps may not only induce
Lmx1a but also induce co-factors required to make neural progenitors
competent to Lmx1a roof plate inducing activity. An alternative hypothesis is
that Bmps can induce roof plate via Lmx1a-independent mechanisms.
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Discussion |
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Lmx1a initiates a regulatory cascade that leads to formation of functional roof plate being a major mediator of early Bmp signaling in dorsal spinal cord
Our previous analysis of the dreher mouse established that
Lmx1a is necessary for roof plate development
(Millonig et al., 2000). In
the current study, we have demonstrated that Lmx1a is sufficient for
inducing ectopic roof plate when overexpressed in developing chick spinal cord
and in neural explants in vitro. We have demonstrated functional activity of
this ectopic roof plate by showing that it can non-autonomously induce dI1 and
dI3 dorsal interneurons at ectopic positions and suppress specification of dI2
dorsal interneurons in chick developing spinal cord.
In this study we have shown that Lmx1a can induce the expression
of members of the Bmp and Wnt families proteins, which are known to be
important components of roof plate signaling in developing spinal cord
(Liem et al., 1997;
Lee et al., 1998
;
Muroyama et al., 2002
). We
have also conducted analysis of dorsally expressed genes in the
dreher mouse and determined that loss of Lmx1a completely
abolishes Bmp expression in embryonic spinal cord, while Wnt expression is
maintained (Millen et al.,
2004
). These data identify Bmps as important components of
Lmx1a-depedndent roof plate signaling. Also they indicate existence of other,
Lmx1a-independent mechanisms that support Wnt expression in developing spinal
cord and that Wnt signaling may account for residual dI1 interneurons
generated in the dreher spinal cord.
Using an inhibitor approach, we have shown that Lmx1a activation and early roof plate development is dependent on Bmp signaling and not affected when Wnt signaling is downregulated. Surprisingly, when Bmp signaling is ectopically activated, the domain of roof plate induction expands to include almost the entire dorsoventral axis of the spinal cord. This is in contrast to Lmx1a, which can induce roof plate only in the most dorsal region of the neural tube. Importantly, our dreher mouse in-vitro explant experiment data indicate, however, that Bmp4 cannot induce roof plate in the absence of Lmx1a. Thus, Lmx1a must be major mediator of the roof plate inducing activity of Bmp in the developing spinal cord. These data also support the conclusion that in addition to Lmx1a Bmps induce other factors that regulate the extent of roof plate induction.
The phenotype that we observed upon activation of Bmp signaling is
different from the findings of Timmer et al.
(2002). In particular, we
observed roof plate induction when we expressed exogenous Bmp in chick stage
10 neural plates, while Timmer et al. found induction of dI1 interneurons when
ectopic Bmp signaling was activated at later stages, 14-16. Taken together,
these experiments indicate that timing of Bmp activation is critical in dorsal
cell fate decisions, providing in-vivo evidence for the model previously
proposed in vitro (Liem et al.,
1997
).
Lmx1a couples the program regulating cell cycle withdrawal and the program controlling cell fate specification during roof plate development
Building a CNS involves the generation of different neuronal and glial cell
types in correct numbers and at appropriate positions. Numerous studies have
demonstrated that this is achieved by the activation of programs that commit
neural progenitors to cell cycle exit and differentiation, and of programs
directing cellular subtype identity. Recent studies suggest that during
neurogenesis the action of specific proteins, including the products of bHLH
proneural genes, is required to couple these two programs
(Bertrand et al., 2002). Our
data show that Lmx1a is sufficient not only for specifying roof plate fate
when expressed in chick and mouse neural progenitors but also for promoting
the arrest of their division and therefore playing a comparable role during
roof plate development. Interestingly, unlike roof plate competence, neural
progenitors are competent to the cell-cycle withdrawal activity of Lmx1a
independent of their location along the dorsoventral axis of the spinal cord.
Indeed, differentiated cells expressing exogenous Lmx1a within these regions
still express appropriate neuronal subtype identity markers. This clearly
indicates that different activities of Lmx1a, probably executed through
cooperation with different partners, are required for the activation of the
program regulating proliferation of the dorsal progenitors and the program
responsible for the acquisition of the roof plate fate.
The role of Lmx1a in segregation of roof plate cells from neural crest and dI1 dorsal interneuron lineages
Roof plate cells are generated from uncommitted progenitors in the neural
folds and early neural tube that also give rise to neural crest cells and dI1
interneuron precursors (Bronner-Fraser and
Fraser, 1988; Echelard et al.,
1994
; Liem et al.,
1997
; Lee and Jessell,
1999
; Helms and Johnson,
2003
). We show that Lmx1a is specifically expressed by roof plate
progenitors and differentiated roof plate cells. Surprisingly, however,
overexpression of Lmx1a was not sufficient to alter the specification
of neural crest cells either in vivo or in vitro. dreher mice have
been reported to have some defects in a small number of neural crest
derivatives, including abnormal pigmentation and improper formation of spinous
processes of dorsal vertebrae (Manzanares
et al., 2000
) (this study). Although dreher mice lack a
roof plate at all axial levels of the spinal cord, neural crest phenotypes are
restricted to the posterior thoracic region, a small domain along the
anteriorposterior axis of the spinal cord. Using multiple molecular
markers, we found that early neural crest development is essentially normal
along the developing spinal cord of drJ/drJ
embryos, even in the affected region. Thus, abnormal pigmentation and improper
fusion of the vertebra detected in the thoracic region of the dreher
mice are probably late secondary defects, caused by loss of roof plate
signaling rather then direct involvement of Lmx1a in neural crest generation.
Taken together, our data suggest that Lmx1a is not directly involved in neural
crest development. Further studies are necessary to dissect the mechanisms
involved in segregation of roof plate and neural crest lineages.
Importantly, our data indicate that Lmx1a is both necessary and sufficient
for segregating roof plate and dI1 dorsal interneuron progenitors. Thus, our
experiments reveal a dual role for Lmx1a in dI1 dorsal interneuron formation.
First, Lmx1a induces dI1 dorsal interneurons through non-autonomous roof plate
signaling. At the same time, it cell-autonomously inhibits expression of Math1
(Cath1), preventing dorsal midline cells from adopting dI1 dorsal
interneuronal fate. Cross-inhibitory regulation has recently been shown to
play a key role in boundary formation between different classes of dorsal
interneuron progenitors (Gowan et al.,
2001). Our data show that negative regulation of Math1 expression
by Lmx1a plays a similar role in boundary formation between the dI1 dorsal
interneuron domain and the roof plate.
In the current study, we have demonstrated that Lmx1a controls multiple steps of dorsal spinal cord development. Elucidation of the downstream components of the Lmx1a regulatory network will be critical to further understanding of the molecular mechanisms underlying the diverse actions of Lmx1a as an important regulator of dorsal CNS patterning.
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ACKNOWLEDGMENTS |
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Footnotes |
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