1 Mammalian Neurogenetics Group, Center for Childhood Communication, 712
Abramsom Research Center, The Children's Hospital of Philadelphia, 34th and
Civic Center Boulevard, Philadelphia, PA 19104, USA
2 Neuroscience Graduate Group, University of Pennsylvania School of Medicine,
Philadelphia, PA 19104, USA
3 Department of Otorhinolaryngology, University of Pennsylvania School of
Medicine, Philadelphia, PA 19104, USA
4 Laboratory of Reproductive and Developmental Toxicology, National Institutes
of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
5 Department of Orthopaedic Surgery, Department of Molecular, Cellular and
Developmental Biology and Biological Chemistry, David Geffen School of
Medicine at UCLA, Los Angeles, CA 90095, USA
* Author for correspondence (e-mail: crenshaw{at}email.chop.edu)
Accepted 29 July 2004
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SUMMARY |
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Key words: Bone morphogenetic protein receptor type 1, Cre-mediated conditional Bmpr1a knockout, Bmpr1b mutant, Dorsal interneuron development, Neural tube patterning, Mouse
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Introduction |
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Roof plate-derived signals establish regional identities in the dorsal
neural progenitors of the ventricular zone by inducing expression of proneural
basic helix loop helix (bHLH) factors Math1 (Atoh1 Mouse Genome
Informatics), Ngn1/2 and Mash1 (Ascl1)
(Timmer et al., 2002;
Gowan et al., 2001
). Although
the mechanisms responsible for establishing the restrictive pattern of bHLH
factor expression in the ventricular zone are largely unknown, gene knockout
studies have shown that specific bHLH factors are crucial for the formation of
subsets of neurons (Gowan et al.,
2001
). Following exit from the cell cycle, neural progenitors
migrate out of the ventricular zone and begin to differentiate. At this time,
combinatorial expression of homeodomain transcription factors specifies the
emerging populations of interneurons (Thor
et al., 1999
). To date, six populations of neurons have been
characterized that are born within the dorsal murine neural tube between 10
and 12.5 dpc. The six populations can be divided into two classes: class A and
class B neurons. Class A neurons, DI1-DI3, give rise to commissural and other
interneurons of the deep dorsal horn
(Muller et al., 2002
), and
their generation is largely roof plate dependent
(Lee et al., 2000
;
Millonig et al., 2000
).
Generation of class B neurons, DI4-DI6, appear to be roof plate-independent
and require Lbx1 (Gross et al.,
2002
; Muller et al.,
2002
). Although the endogenous mechanisms required for the
formation of these distinct classes are still unclear, BMP signaling regulates
markers of the class A neurons in a gradient-dependent fashion
(Timmer et al., 2002
;
Panchision et al., 2001
).
Thus, BMPs appear to mediate both the progenitor populations and the ultimate
specification of dorsal cell types.
Multiple BMP family members are expressed in the roof plate and epidermal
ectoderm, and in vitro work has suggested that the dorsalizing function of the
roof plate is carried by the BMP signal
(Liem et al., 1997). The BMPs
are members of the TGFß superfamily of cell signaling molecules that play
many important roles throughout embryogenesis (for a review, see
Hogan, 1996
) and in nervous
system development (for reviews, see
Mehler et al., 1997
;
Ebendal et al., 1998
). BMP
ligands bind to transmembrane serine-threonine kinase receptors. The receptors
are composed of type 1 and type 2 subunits, of which there are multiple
subtypes for each component (for reviews, see
Derynck and Zhang, 2003
;
Massague, 1996
;
Massague, 1998
). Type 1 and
type 2 receptors alone exhibit low-affinity binding, while in combination,
high-affinity binding can be achieved. Cooperative binding of ligands to the
oligomeric receptor complex leads to phosphorylation of the type 1 component
by the type 2 kinase domain. Ligand binding initiates the downstream effects
of BMP signaling, namely phosphorylation of SMAD proteins by the type 1
receptor subunit. Translocation of phosphorylated SMAD to the nucleus directs
the downstream effects of BMP signaling. Multiple subtypes of BMP receptors
are found in the developing neural tube. At the time of critical events for
cell type specification, the predominant type 1 receptors are BMPR1A and
BMPR1B (for a review, see Ebendal et al.,
1998
). Because of the multiple BMP family members expressed by the
neural tube during development, components of the BMP signaling cascade are
excellent targets for manipulation in assessing the roles of BMPs during
nervous system development.
BMPs and other TGFß family members mimic effects of roof plate tissue,
while BMP antagonists are inhibitory for dorsal neural marker expression
(Liem et al., 1997). More
recently, in vivo manipulation of BMP signaling has suggested that these
pathways are crucial for dorsal interneuron populations
(Nguyen et al., 2000
;
Panchision et al., 2001
;
Timmer et al., 2002
).
Loss-of-function analyses in the mouse have for technical reasons provided
little insight into endogenous BMP activity in specification of dorsal cell
fate (for a review, see Chang et al.,
2002
). Functional redundancy of BMP proteins and early lethality
of BMP signaling mutations have prevented traditional knockout studies from
establishing the role of BMPs in neural tube patterning. A role for a
BMP-related protein in dorsal neural tube development has been shown, as loss
of Gdf7 expression leads to a specific loss of DI1 interneurons
(Lee et al., 1998
). However,
the DI1 cells are initially specified in Gdf7-null mice, but are
subsequently lost suggesting that BMP activity may also play an important role
in maintaining cells of the dorsal neural tube. Thus, clear genetic evidence
for a role of BMP signaling in dorsal cell fate determination and maintenance
is still lacking.
Here, we describe our analysis of the role of BMP signaling in development
of dorsal cell phenotypes in the neural tube using conditional and classic
knockout approaches to disrupt BMP signaling. We have generated a double
knockout of BMP type 1 receptors in the neural tube by using a conditional
knockout of Bmpr1a (Ahn et al.,
2001; Mishina et al.,
2002
) and a classic knockout of Bmpr1b
(Yi et al., 2000
). Either
mutation alone does not abrogate the BMP signal nor yield a dorsal neural tube
patterning defect. By contrast, the double knockout eliminates BMP signaling
in the neural tube during the period of cell type specification. We
demonstrate that loss of BMP signaling in the neural tube leads to disruption
of the dorsal cell populations. Specifically, Math1-expressing
progenitors are lost with a subsequent loss of DI1 interneurons. In addition,
a profound reduction in DI2 neurons is observed in double mutant animals.
Abrogation of the BMP signal also effects other signaling pathways within the
neural tube as seen by changes in the expression of Wnts and Ids, indicating a
complex network of interactions is probably responsible for proper dorsal cell
specification. In this paper, we provide direct genetic evidence that BMP
signaling is necessary for the development of dorsal populations in the
developing spinal cord in the mouse.
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Materials and methods |
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In situ hybridization
In situ hybridization analyses were carried out using 20-25 µm
cryosections of embryos fixed overnight in 4% paraformaldehyde (PFA) as
described previously (Ahn et al.,
2001). The following mouse probes were used: En1 (A.
McMahon), Foxd3 (P. Labosky), Lhx2 (J. Botas), Lhx9
(H. Westphal), Lmx1b (R. Johnson), Math1 (J. Johnson),
Mash1 (D. Anderson), Ngn2 (D. Anderson), Wnt1 (A.
McMahon) and Wnt3a (A. McMahon). Images were taken on a Leica DM-IRBE
inverted microscope using a Leica DC500 digital imaging system.
Immunohistochemistry, immunofluoresence and microscopy
Phosphorylated SMAD1 (phospho-SMAD1) immunohistochemistry was performed by
modification of previously published methods
(Ahn et al., 2001). Briefly, 20
µm cryosections were processed for tyramide amplification (TSA Indirect
Tyramide Signal Amplification Kit, Perkin Elmer Life Science) and
immunoperoxidase labeling (Vectastain ABC Kit, Vector Labs). Antigen unmasking
was performed by heating slides in 10 mM sodium citrate pH 9.0 in an 80°C
water bath for 30 minutes. The slides were incubated overnight at 4°C in a
1:10,000 dilution of anti-phospho-SMAD1 (Cell Signaling Technology) in 5%
normal goat serum.
Immunofluorescence was performed on 14 µm cryosections using embryos fixed in 4% PFA for 30 minutes. Slides were fixed in cold acetone (20°C), washed and blocked for 1 hour in blocking solution containing 10% fetal calf serum, 0.5% Triton X-100. Sections were incubated overnight at 4°C in primary antibody diluted in blocking solution. Sections were then washed and incubated with a biotinylated secondary (diluted in blocking solution) at room temperature. After further washes, slides were incubated with a fluorochrome-conjugated avidin. Staining with mouse primary antibodies was accomplished with the M.O.M. Kit (Vector Labs). The following antibodies were used: Pax2 (Zymed), Msx2 (4G1), Isl1 (39.4D5), Lim1/2 (4F2), Pax6 and Pax7 (Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences).
TUNEL analyses were accomplished using 14 µm cryosections and processed
according to published protocols (Grinspan
et al., 1998). Assays for cell proliferation were carried out by
immunolabeling for the mitosis marker, phosphorylated histone H3
(Hendzel et al., 1997
;
Nowak and Corces, 2000
).
Cryosections (14 µm) were washed, incubated in 0.5% Triton and blocked in
5% normal goat serum (Vector laboratories). Slides were incubated overnight at
4°C with anti-phospho-histone H3 antibody (Upstate Biotechnology; 1:250).
Slides were washed and incubated with secondary antibody (Jackson
Laboratories) and nuclei were visualized using
4',6-diamidino-2-phenylindole (DAPI, Sigma) staining.
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Results |
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The spatial and temporal expression of the Bcre32 transgene was
determined in the neural tube by crossing the Bcre32 strain with the
ROSA reporter strain (Soriano,
1999). The resulting Bcre32-driven expression of
lacZ is demonstrated in Fig.
2. Expression is first detected in the anterior neural folds at
8.5 dpc (Fig. 2A) and
progresses caudally. As seen in Fig.
2B,D, the entire neural ectoderm of the rostral spinal cord
demonstrates Bcre32-mediated lacZ expression by 9.75 dpc. By
10.5 dpc, lacZ expression is detected throughout the neural tube
(Fig. 2C).
|
BMP signaling eliminated in Bmpr1a and Bmpr 1b double mutant animals
The loss of BMP receptor signaling was directly assayed by examining the
phosphorylation of SMAD1, which is phosphorylated by signaling through the BMP
type 1 receptor subunits, BMPR1A and BMPR1B. At 10.0 dpc, high levels of
phosphorylated SMAD1 (phospho-SMAD1) immunoreactivity are seen in the dorsal
neural ectoderm of normal animals (Fig.
3A). We do not detect any changes in phospho-SMAD1 immunostaining
in either the neural tube of Bmpr1a conditional knockouts or
Bmpr1b mutant animals (data not shown), suggesting that abrogation of
BMP signaling requires the loss of both type 1 BMP receptors. Double mutant
animals show a complete loss of immunopositive cells in the dorsal neural
tube, except in the specialized tissue of the roof plate where a few
phospho-SMAD1-labeled cells remain (arrow,
Fig. 3B). Expression in neural
crest cells is unaffected (Fig.
3B). These data demonstrate that Bmpr1a and
Bmpr1b are functionally redundant for the phosphorylation of SMAD1 in
the dorsal neural tube.
|
BMP signaling is required for development of the DI1 population of sensory interneurons in the dorsal neural tube
The effects of BMP signaling loss in the neural tube were studied by
examining the expression of proneural bHLH transcription factors. Math1,
Ngn2 and Mash1 expression marks distinct populations of neuronal
progenitors in the developing neural tube
(Gowan et al., 2001). The most
dorsal population of Math1-expressing neural progenitors gives rise
to the DI1 population of sensory interneurons
(Bermingham et al., 2001
).
Although Math1 expression is detected at 10.0 dpc, by 10.5 dpc double
mutant animals demonstrate a complete loss of Math1 expression (data
not shown, Fig. 4A,B). The loss
of BMP signaling also results in a dorsal shift of the dorsal precursor
populations expressing Ngn2 and Mash1
(Fig. 4). Ngn2-expressing cells now occupy the most dorsal aspect of the neural
tube, adjacent to the roof plate (Fig.
4C,D). Although Mash1 expression is also shifted, the
broad region of Mash1 expression remains intact in the mutant animals
(Fig. 4E,F). Ngn2 also
marks a broad domain of ventral neuronal precursors and this expression
remains unaffected in double mutant animals
(Fig. 4C,D).
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BMP signaling is important for specification of DI2 interneurons but not mid-dorsal or ventral populations
The neurons that arise from the next most dorsal aspect of the neural tube,
the DI2 population, is another population of ventrally migrating sensory
interneurons (for a review, see Helms and
Johnson, 2003). The DI2 neurons express markers for the LIM
homeodomain factor, Lim1/2 and the winged-helix factor, Foxd3. To determine
whether the DI2 population of sensory interneurons was affected in the double
mutant animals, we examined the expression patterns of these markers. Dorsal
Foxd3 expression is markedly decreased, while ventral Foxd3
expression is intact (Fig.
6A,B,E,F). In our mutant animals, a few cells in the dorsal-most
aspect of the neural tube retain expression of Foxd3
(Fig. 6E,F), and these are
located more dorsally than normal, adjacent to the roof plate, in the region
that is normally occupied by DI1 interneurons
(Fig. 6E,F).
We further characterized the loss of DI2 neurons by examining the
expression of Lim1/2. Lim1/2 is expressed in multiple regions, marking three
distinct dorsal populations: DI2, DI4 and DI6 (for a review, see
Helms and Johnson, 2003;
Gross et al., 2002
;
Muller et al., 2002
). DI4 and
DI6 populations additionally express Pax2. Therefore, Lim1/2-positive,
Pax2-negative immunolabeling is indicative of the DI2 population, while
Lim1/2-positive, Pax2-positive staining marks the mid-dorsal populations
(Fig. 6C). In our double mutant
animals, very few Lim1/2-positive, Pax2-negative cells are observed
(Fig. 6D,1). In addition, those
that are seen are located in the dorsal-most region of the neural tube,
adjacent to the roof plate. This further indicates the loss of the DI2
population and a dorsal shift to areas normally expressing markers of DI1
neurons.
To further understand the effect of BMP signaling on development of dorsal cell types, we examined the DI3 and DI4 populations, located just ventral to the DI2 cells. DI3 cells are marked by expression of Isl1/2. In double mutant animals, Isl1/2-positive cells are shifted, such that some cells are found almost adjacent to the roof plate (arrowhead, Fig. 6H). DI4 cells are marked by the expression of Pax2, and are intermingled with, yet distinct from, DI3 cells, as demonstrated by no co-labeled cells (Fig. 6G,H). The dorsal shift in the DI4 population is also seen in Fig. 6D as the Lim1/2 positive, Pax2-positive immunoreactive cells are more dorsally located. Thus, it is evident that the abrogation of the BMP signal leads to a loss of DI1 and DI2 sensory interneurons, and an associated dorsal expansion of DI3 and DI4 populations. The dorsal expansion is also accompanied by an increase in the number of cells expressing markers for DI3 and DI4 neurons (Fig. 6I).
The observed expansion of these populations is not accompanied by changes in proliferation. Immunostaining for the mitosis marker phospho-histone H3 in normal neural tubes demonstrates that cells immediately adjacent to the central canal of the spinal cord were undergoing or had recently undergone DNA replication at the time of embryo harvesting. This pattern of proliferation is intact in the mutant animals (Fig. 6G,H). At 10.5 dpc, there are no quantitative differences in the proliferation of neuronal progenitors between normal, single mutant and double mutant animals (Fig. 6G,H; data not shown). Additionally, no differences in cell proliferation were detected at 10.25 or 11.5 dpc (data not shown). Thus, BMP signaling does not appear to regulate neuronal cell proliferation in the developing spinal cord.
Neurons derived from the more ventral regions of the dorsal neural
progenitor domains migrate to occupy the upper layers of the dorsal horn
(Muller et al., 2002). Further
classes of sensory interneurons, DI5 and DI6, are born from
Mash1-expressing progenitors cells of the dorsal spinal cord. Lmx1b
exclusively marks the population of DI5 neurons. As seen in
Fig. 7B, Lmx1b
expression is normal in the double mutant animals. DI6 neurons express the
same homeodomain profile as DI4 neurons, but arise at a distinct location
along the dorsoventral axis. DI6 interneurons in our mutant animals appear to
be intact as well, as indicated by the expression of Lim1/2
(Fig. 7D). Thus, it is
evidenced that mid-dorsal populations of neurons DI3-DI6 are generated
independently of BMP signaling in the dorsal neural tube.
|
Wnt expression is downregulated in Bmpr double mutant animals
In addition to the BMP family members, two members of the Wnt family,
Wnt1 and Wnt3a, are expressed by roof-plate cells throughout
the time of neurogenesis (for reviews, see
Hollyday et al., 1995;
Parr and McMahon, 1994
). To
examine the effects of BMP signaling abrogation on Wnt signaling in the dorsal
neural tube, we examined the expression patterns of Wnt1 and
Wnt3a in our double mutant animals. As seen in
Fig. 8, the roof plate and
adjacent dorsal neuroepithelium at 10.5 dpc in normal animals express
Wnt1 and Wnt3a. The loss of BMP signaling in our mutants
leads to a decreased domain of Wnt expression. Wnt1 and
Wnt3a become restricted to the roof plate in mutant animals with a
complete loss of expression in the adjacent tissue
(Fig. 8B,D). This effect is
seen as early as 10.25 dpc (data not shown). Again, this loss is not
accompanied by changes in cell proliferation in mutant animals
(Fig. 6G,H). Thus, the loss of
BMP signaling reduces, but does not eliminate, Wnt signaling in the developing
spinal cord.
|
Id1 and Id3 are expressed in the neural progenitors adjacent to the roof plate at 10.0 dpc in normal animals (Fig. 9A,E). By contrast, Id2 shows a low level of expression throughout the progenitor domain of the dorsal spinal cord with higher expression in the dorsal midline (Fig. 9C). In our mutant animals, Id1 and Id3 are greatly reduced at 10.0 dpc (Fig. 9B,F). The dorsal expression is lost by 10.5 dpc (Fig, 9G,H, data not shown). Ventral Id1 and Id3 expression is intact (arrowhead, Fig. 9B,F). Id2 expression appears unaffected at 10.0 dpc, but is lost dorsally by 10.5 dpc (Fig. 9C,D; data not shown). Thus, the expression of Id family members is affected by the loss of BMP signaling in the Bmpr1a;Bmpr1b double mutant animals.
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Discussion |
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Genetic ablation of the roof plate, as seen in the dreher mutant
mice, also leads to a decrease in DI1 populations
(Millonig et al., 2000).
However, the progenitor population is intact, suggesting that the BMP signal
that is crucial for maintaining progenitor populations may be extrinsic to the
roof plate, possibly from earlier BMP activity. Although our studies do not
elucidate the mechanisms of initial establishment of the dorsal progenitor
domains, we clearly show that BMP signaling is directly or indirectly
necessary for maintenance of the D1 precursor cells. However, proper
specification of the differentiated DI1 cells requires BMP signaling, as seen
in our studies. Moreover, Math1 is necessary, but not sufficient, for DI1
differentiation (Gowan et al.,
2001
), further suggesting that BMPs, in addition to Math1, may be
necessary for specification of the postmitotic DI1 population. In the absence
of Math1 expression, DI1 precursors neither differentiate nor migrate
(Bermingham et al., 2001
).
Thus, in our mutant animals, it is not possible to determine whether the
observed loss of Lhx2- and Lhx9-expressing cells is only
secondary to the loss of Math1 expression. Further studies will be
needed to determine if loss of BMP signaling in the presence of normal Math1
activity also affects DI1 specification. Nonetheless, our studies, in
conjunction with previous work, indicate that BMP signaling is necessary for
expression and maintenance of the D1 precursor.
Our analyses demonstrate that other dorsal progenitor domains remain intact
in double knockout, as seen by the continued expression of Ngn2 and
Mash1. This suggests that BMP signaling, at this stage, is not
necessary for establishment of the remaining dorsal ventricular zone areas.
Toxin-mediated roof plate ablation does affect the D2 precursors, as shown by
loss of Ngn1 expression (Lee et
al., 2000). These results, taken with ours, suggest that other
factors originating from the roof plate may be essential for formation of D2
precursor populations. Our mutant animals demonstrate that the formation of
DI2 interneurons is affected by loss of BMP signaling, indicating that
specification of postmitotic DI2 cells is BMP dependent.
The bHLH proteins, Math1, Mash1, Ngn1 and Ngn2 establish and maintain the
ventricular zone regions necessary for correct specification of neuronal fates
(for a review, see Gowan et al.,
2001; Ross et al.,
2003
). Id proteins, negative regulators of bHLH proteins, are
expressed within the ventricular zone of the developing neural tube. Members
of the Id protein family have been implicated as crucial for maintenance of
neural progenitor populations by antagonizing proneural bHLH factors, and
downregulation of Ids is hypothesized to be a crucial molecular switch for
terminal differentiation of neural precursors cells (for reviews, see
Norton, 2000
;
Ross et al., 2003
). Id1,
Id2 and Id3 expression has been shown to be directly induced by
BMP activity in a variety of cell lines
(Hollnagel et al., 1999
).
Thus, Ids are candidates for mediators of the actions of BMPs in neural
patterning. Although we have observed alterations in Id expression in
mutant animals (Fig. 9), these
changes do not appear to be profound enough to fully account for the observed
patterning defects in our mutant animals. Some Id expression remains
when losses of neural markers have already been detected. In addition, we
suggest that the dorsal patterning defects seen in our double mutants are
independent of the Id expression changes, as Id1;Id3 double
knockout animals show expanded Math1 expression
(Lyden et al., 1999
).
In contrast to the DI1 and DI2 cell populations, we observed an expansion
of DI3 and DI4 cells in the double knockout animals. Toxin-mediated roof-plate
ablation leads to loss of DI3 interneurons
(Lee et al., 2000). Thus,
roof-plate signals other than BMPs are likely to be important. The increased
number of DI3 and DI4 cells suggests that these cells have switched from a
more dorsal fate in the absence of BMP signaling, although the absolute number
of cells lost can not be fully accounted for by the DI3 and DI4 expansion.
Other dorsal and ventral populations are unchanged, consistent with other
reports that class B neurons and ventral populations are roof plate
independent (Gross et al.,
2002
; Muller et al.,
2002
).
The Bmpr1a conditional knockout and the Bmpr1b null
animals alone do not show any of the neural tube defects observed in the
double mutant animals. Therefore, our studies demonstrate that, in the mouse,
these two BMP type 1 receptor subtypes are functionally redundant in dorsal
patterning of the neural tube. This differs from the results obtained by
Panchision et al. (Panchision et al.,
2001), who observed sequential and differential actions of
Bmpr1a and Bmpr1b activated constructs in transgenic
animals. The contradictory nature of our findings suggests that the use of
constitutively activated receptors in this context does not accurately mimic
endogenous roles of the BMP type 1 receptors. However, both reports do show a
clear role for BMPs in dorsal cell type specification. Their report also
suggests a role for BMP signaling in proliferation and apoptosis. Again, we do
not see such differences in double knockout animals. Furthermore, BMPs
probably activate proliferation through observed induction of Wnt signaling,
and in our animals we have some remaining Wnt expression.
BMPs and Wnts may act cooperatively in dorsal cell type specification
It is likely that other non-TGFß related proteins are involved in
dorsal spinal cord development. Such candidates for these BMP-independent roof
plate activities are the Wnt family members. There are many examples
throughout embryogenesis of the important interactions between Wnt and BMP
family members (Ahn et al.,
2001; Roelink,
1996
; Soshnikova et al.,
2003
) and Wnts have been shown to be directly inducible by BMP
signaling (for reviews, see Roelink,
1996
; Panchision et al.,
2001
). We see some initial expression of Wnt1 and
Wnt3a, with subsequent loss of expression in the dorsal neural
ectoderm (Fig. 8, data not
shown). However, roof plate expression remains intact. It is thus possible
that BMP signaling could induce and/or maintain Wnt expression
specifically in the dorsal neural ectoderm. Alternatively, the loss of Wnt
expression could be secondary to the general perturbation of cell
specification seen in the double mutant animals.
The role of Wnts in dorsal cell specification is still not completely
understood. Wnt1;Wnt3a double knockouts demonstrate losses in D1 and
D2 precursor and decreased DI1 and DI3 cells without accompanying alterations
in BMP expression (Muroyama et al.,
2002). These defects are similar to those seen in the roof plate
ablation studies (Lee et al.,
2000
). The losses seen in the Wnt double knockouts are, however,
not complete, suggesting that Wnts and BMPs may act cooperatively for dorsal
cell specification. Furthermore, Wnt3a-conditioned media induced expression of
DI1 and DI3 markers (Muroyama et al.,
2002
). It is thus possible that a Wnt gradient works in
conjunction with or is downstream of BMP signaling to specify dorsal cell
types. However, Wnt signaling through the ß-catenin pathway has been
shown to regulate cell cycle progression and negatively regulate cell cycle
exit in neural cells (Chenn and Walsh,
2002
; Megason and McMahon,
2002
; Soshnikova et al.,
2003
; Zechner et al.,
2003
). Bmpr double knockouts do not show any alterations
in cell proliferation despite the perturbation in Wnt expression. It is thus
likely that either the changes we see in Wnt expression are too late to alter
cell proliferation or that the residual expression in the roof plate is
sufficient to maintain the mitogenic effects of Wnts.
In conclusion, we have demonstrated that signaling through the predominant BMP type 1 receptors, Bmpr1a and Bmpr1b, is crucial for maintenance of dorsal progenitor cells and for the specification of dorsal neural cell types. As summarized in Fig. 10, the double knockout animals demonstrate losses of DI1 and DI2 sensory interneurons and their accompanying molecular markers. The role of BMPs in the neural tube is complex, as other signaling pathways, such as Wnts and Ids, are also affected. Further work is needed to understand fully the complexity of these interactions and their role in neural tube patterning.
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ACKNOWLEDGMENTS |
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