1 Instituto de Biología Molecular de Barcelona (CSIC), Parc Cientific de
Barcelona, C/Josep Samitier 1-5, Barcelona 08028, Spain
2 Instituto de Investigaciones Biomédicas de Barcelona (IDIBAPS-CSIC),
C/Rosselló 161, Barcelona 08036, Spain
Authors for correspondence (e-mail:
emgbmc{at}ibmb.csic.es
and
spfnqi{at}iibb.csic.es)
Accepted 23 March 2004
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SUMMARY |
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Key words: Cerebellar development, Granule neuron, Sonic hedgehog, Bone morphogenetic proteins, Smad proteins, Mouse, Chick
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Introduction |
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Recent studies have provided insights into the molecular nature of the
signals directing the subsequent steps of cerebellar cortex development.
Dorsal midline-derived bone morphogenetic proteins (BMPs), which are members
of the transforming growth factor ß (TGFß) superfamily, appear to
act on a regionalized cerebellar anlage to induce the generation of granule
neuron progenitors that migrate from the rhombic lip to populate the EGL
(Alder et al., 1999).
Furthermore, sonic hedgehog (Shh) within the EGL and secreted from adjacent
Purkinje neurones acts as a potent mitogenic signal to expand the granule cell
progenitor population (Dahmane and Ruiz i
Altaba, 1999
; Kenney and Rowitch, 1999;
Pons et al., 2001
;
Wallace, 1999
;
Wechsler-Reya and Scott,
1999
). Subsequent steps of granule cell differentiation require
exit from the cell cycle, initiation of differentiation and migration through
the Purkinje cell layer, events that occur within a Shh-rich environment.
Therefore, termination of granule cell proliferation is probably not due to
reduced exposure to Shh, rather it is likely to result from signals that can
suppress the proliferative response to Shh. Extracellular matrix glycoproteins
(Pons et al., 2001
) and
fibroblast growth factors (FGFs)
(Wechsler-Reya and Scott,
1999
; Pons et al.,
2001
) are able to differentially modulate but not to totally
suppress Shh-mediated proliferation of granule cell precursors, making them
unlikely candidates to account completely for the suppression of the
proliferative response. However, hedgehog proteins and BMPs are co-expressed
at many sites of cell-cell interaction during development
(Bitgood and McMahon, 1995
) and
are known to have opposing activities in many developmental paradigms
(Lee and Jessell, 1999
;
Mekki-Dauriac et al., 2002
;
Patten and Placzek, 2002
;
Zhu et al., 1999
). We have
therefore undertaken an analysis of BMP protein expression and function in the
developing cerebellum, as putative antagonists of Shh-mediated
proliferation.
Shh is a secreted protein that signals through its receptor patched
(Ptch1), an eleven-pass transmembrane receptor. In the absence of Shh, Ptch1
associates with and sequesters the activity of smoothened (Smo) (for reviews,
see Ingham and McMahon, 2001;
Ho and Scott, 2002
;
Nybakken and Perrimon, 2002
;
Martí and Bovolenta,
2002
). In response to the binding of Shh, Ptch1 releases Smo
inhibition, which then activates a G
i subunit to inhibit cAMP
production within the cell. The Gli family of zinc-finger transcription
factors act at the last known step in the Shh signal-transduction pathway
(reviewed by Ruiz i Altaba et al.,
2002a
; Ruiz i Altaba et al.,
2002b
). Within the Shh-receiving cell, Gli proteins are regulated
in the cytoplasm via multiple distinct molecular mechanisms. The cyclic
AMP-dependent protein kinase (PKA) acts as a common negative regulator such
that Gli repressor forms are generated by PKA-mediated phosphorylation and
that inhibition of PKA activity releases Gli activated forms. Gli proteins
then move to the nucleus where, by acting together with co-activators
(Goodman and Smolik, 2000
) or
with co-repressors (Dai et al.,
2002
), they regulate transcription of target genes.
BMPs are also secreted proteins that use a relatively simple mechanism to
signal to the nucleus (for reviews, see
Massagué, 2000;
Massagué et al., 2000
;
Shi and Massagué,
2003
). BMP ligands bring together members from two families of
receptor serine/threonine kinases, known as the type I and type II receptors.
Type II receptors activate type I receptors that then propagate the signal by
phosphorylating Smads, which are the only known BMP receptor substrates
capable of signal transduction. Phosphorylation causes Smads to move to the
nucleus where they assemble complexes that directly control gene expression.
Each different ligand may have a choice of several type I and type II
receptors, and a given cell may express different receptor forms; however, in
the case of BMPs, the various type I receptors funnel their activities through
one of three different Smads (Smad1 or the closely related Smad5 and Smad8).
Phosphorylation of Smad1/5/8 increases their affinity for a particular member
of the family, Smad4, that functions as a shared partner (co-Smad), and is
required for the assembly of active transcriptional complexes. Activated
Smad1/5/8 moves to the nucleus where, acting together with co-activators
(Goodman and Smolik, 2000
) or
with co-repressors (Wang et al.,
2000
), it activates/represses target genes transcription.
We describe the expression of several BMPs in the developing cerebellar cortex. In search of a putative functional antagonism of Shh activity by BMPs, we show that Bmp2 and Bmp4, but not Bmp7, are able to totally overcome Shh-induced proliferation of granule cell precursors. Furthermore, we show that Bmp2-mediated differentiation of cerebellar granule neurones is mediated by Smad5 signalling and that Smad5 expression is sufficient to trigger granule cell precursor differentiation, thus providing a strong basis for understanding the molecular control of granule neuron proliferation/differentiation.
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Materials and methods |
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Antibodies and chemicals
The monoclonal antibody anti-bromodeoxyuridine (BrdU) was obtained from the
Developmental Studies Hybridoma Bank (Iowa). The monoclonal antibody against a
unique ßIII-Tubulin (Tuj-1) was obtained from MEDPASS (Grand Duché
de Luxembourg) and used to identify postmitotic neurons. The rabbit polyclonal
anti-GFAP (AB1980) was purchased from CHEMICON. The rabbit polyclonal
anti-calbindin-D28K was purchased from Swant. The rabbit polyclonal
anti-phospho-histone 3 (P-H3) was purchased from Upstate Biochemicals.
Anti-Smad1, anti-Smad5 and anti-Smad8 were purchased from Santa Cruz. A phospho-specific antibody that recognizes the activated forms of Smad1/5/8 was purchased from Cell Signaling Technologies.
The E. coli-produced 19 kDa N-terminal fragment of recombinant sonic hedgehog (based on the human sequence) used in this study was a gift from Biogen (Cambridge, MA).
Bmp2 was obtained from Genetic Institute (Cambridge, MA), Bmp4 was purchased from R&D Systems (Minneapolis, MN) and Bmp7 from Creative BioMolecules (Boston, MA).
In situ hybridisation, immunohistochemistry and RT-PCR
In situ hybridisation was performed on 50 µm vibratome sections
following standard procedures. Hybridisation was revealed by alkaline
phosphatase-coupled anti-digoxigenin Fab fragments (Boehringer Mannheim).
The following probes have been previously described: chick sonic
hedgehog (Riddle et al.,
1993); mouse Shh
(Echelard el al., 1993
); chick
bmp2 and bmp4
(Francis-West et al., 1994
);
chick bmp7 (Houston et al.,
1994
); mouse Bmp2, Bmp4 and Bmp7
(Piedra and Ros, 2002
). Chick
Smad1 and Smad5 probes were obtained from Dr Juan
Hurlé (University of Cantabria, Spain), and Smad8 probe was
obtained from the chicken EST project (UK-HGMP RC). Mouse Smad1 and
Smad5 probes were obtained from Dr E. Robertson (Harvard University,
MA).
Immunohistochemistry was performed on free-floating vibratome sections (50 µm) based on standard procedures. After single or double staining, sections were mounted, analysed and photographed using a Leica Confocal microscope.
RNA extractions were done following the user manual of the NucleoSpin RNA purification kit from BD Biosciences. RT-PCRs were performed following the user manual of the Titanium One-Step RT-PCR kit from as BD Biosciences. Primer sequences are available on request.
Primary culture of granule cells
Cerebellar cultures were performed using a modification of the procedure
described by Meyer-Franke et al.
(Meyer-Franke et al., 1995).
Chemicals and incubation times were optimised for the simultaneously
processing of four P6 mouse cerebellae. Cerebellae were aseptically removed,
washed once in Earls Balanced Salt Solution (EBSS) (Invitrogen), cut
into small pieces (1 mm) and transferred to a 50 ml screw cap tube. Tissue
fragments were allowed to settle, the excess EBSS aspirated and 4 ml of EBSS
containing 100 U/ml of DNAse (Worthington, Lake Wood, NJ), 1 mM
CaCl2 and 1 mM MgCl2 was added and gently mixed with
tissue fragments. Finally, 100 U of papain (Worthington) that was
pre-activated for 30 minutes at 37°C in 1 ml of activation buffer (EBSS
containing 5 mM L-Cys, 2 mM EDTA and 0.067 mM ß-mercaptoethanol) was
added, air was displaced with 95%O2/5%CO2 and the sample
was incubated for 90 minutes on a shaking platform at 37°C. At the end of
this period, the tube was vortexed at low speed for 1 minute and undigested
fragments were allowed to settle, the supernatant was then transferred to a
fresh 15 ml screw cup tube and centrifuged at 600 g for 5
minutes. The supernatant was aspirated and the pellet resuspended in 3 ml of
EBSS containing 3 mg of ovomucoid protease inhibitor (Worthington), layered
onto an albumin cushion consisting of 5 ml of EBSS containing ovomucoid
protease inhibitor and ovoalbumin (Worthington) each at 10 mg/ml and
centrifuged at 600 g for 5 minutes. The resulting pellet was
resuspended in neurobasal medium (Invitrogen), and the cell number and
viability was assessed using a haemocytometer. The typical yield from this
protocol is 12-15 million cells per cerebellum and cell viability is higher
than 90%. Cells were plated onto laminin (GIBCO, BRL)-coated tissue culture
dishes or glass coverslips at 100,000 cells/cm2 in neurobasal
medium supplemented with B27 (Invitrogen) containing 20 mM KCl and were
maintained in a humidified incubator at 37°C in a 5% CO2
atmosphere. This culture medium has been optimized to support neuronal
survival and minimize glial proliferation; more than 95% of the cells
displaying neuronal markers after 48 hours in culture. Twelve hours before
harvesting cultures were pulsed-labelled with [3H]thymidine (1
µCi/ml) for incorporation of radioactivity. Cultures plated onto glass
coverslips were fixed in 4% paraformaldehyde and processed for immunostaining.
TUNEL staining was performed using the In Situ Cell Death detection Kit
(Roche).
Organotypic slice culture experiments
Cerebellar slice cultures were prepared either from mouse P4-P6 or from
chick stage 38-40 [embryonic day (E) 12-13] cerebellae. Brains were
aseptically removed and the cerebellae were excised. Tissue pieces were cut
into sagittal slices (50 µm) using a tissue chopper (McIlwain Tissue
Chopper, Vibrotome 800) and maintained in ice cold Earls Balanced Salt
Solution (EBSS) (Invitrogen). Cerebellar slices were placed onto 1 µm
polycarbonate filters (Costar) and the filters were supported by stainless
steel grids on the surface of the culture medium (Dulbeccos Modified
Eagles Medium/F12 supplemented with 2 mM glutamine, penicillin/streptomycin
and B27 (Invitrogen). The medium was changed every 24 hours.
Heparin acrylic beads (Sigma, 80 µm) were used for exogenous application of proteins. Beads were rinsed with PBS and incubated with 5 µl of protein solution at room temperature for 1 hour before use. Beads were soaked either in PBS (control beads) or in recombinant purified human Shh (1 µg/ml), recombinant human Bmp2 (0.1 µg/ml), Bmp4 (0.1 µg/ml) or Bmp7 (0.1 µg/ml). Four hours prior to fixation, cultures were treated with 50 ng/ml BrdU, fixed in 4% paraformaldehyde and processed for immunohistochemistry.
Western blotting
Primary cultures were lysed in 1xSDS loading buffer [10% glycerol, 2%
SDS, 100 mM DTT and 60 mM Tris-HCl (pH 6.8)] and the DNA disrupted by
sonication. Samples were then separated by SDS-PAGE gel electrophoresis and
transferred to nitrocellulose membranes; blocked with 8% non-fat dry milk in
TTBS (150 mM NaCl; 0.05 Tween-20 and 20 mM Tris-HCl pH 7.4) and probed with
the different antibodies used. The blots were developed using anti-rabbit
coupled peroxidase plus the ECL system (Amersham). Quantifications were
performed using a Molecular Dynamics Densitometer.
Transfection of granule cell precursors
After 24 hours in culture, granular cell preparations were transfected
using FuGENE6 reagent (Roche). Briefly, P6 granular cells platted onto PLL/LN
coated cover-slips were grown in neurobasal-B27 containing 3 µg/ml of Shh
for 24 hours. For the transfection, 50 µl of neurobasal media containing 2
µl of FuGENE plus 0.5 µg of DNA vectors were added to each well of a
12-well culture dish. Full coding regions of Smad1, Smad5 and Smad8 were
subcloned in the bicistronic vector pCIG (Megason and McMahon, 2001) that
contains nuclear EGFP. The cultures were allowed to grow for 48 hours in the
same media, fixed in 4% paraformaldehyde and processed for
immunocytochemistry.
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Results |
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Furthermore, cultures treated with Shh together with Bmp2 or Bmp4 showed
the same phenotype as control cultures in which more than 95% of the cells are
ßIII-Tubulin positive and less than 5% cells are GFAP positive
(Fig. 3D). However, cells grown
in the presence of Shh and Bmp7, in which proliferation is still high, show
the presence of groups of undifferentiated cells that are not labelled with
either neural or glial markers (Fig.
3E), similar to the phenotype of cultures treated with Shh alone
(Fig. 2C). A moderate
phenotypic change occurs in glial cells grown in the presence of DBA, either
alone or together with Shh, in which astrocyte processes are longer and
thinner than controls (data not quantified,
Fig. 3F,G). The role of Bmp2
and Bmp4 in astrocytic differentiation has been well characterised both in
vitro (Angley et al., 2003;
Nakashima et al., 2001
;
Zhu et al., 1999
) and in vivo
(Gomes et al., 2003
) and,
consistent with this role, in our culture conditions we observe the presence
of progenitor cells expressing GFAP in cultures grown in the presence of Bmp2
or Bmp4 alone (Fig. 3H), which
are more abundant in cultures treated with Shh and Bmp2/4
(Fig. 3I).
We next tested the effect of Bmp2 treatment on components of the Shh
pathway. Cultures were grown for 48 hours in the presence of Shh, Bmp2 (200
ng/ml) was then added for different time points
(Fig. 3J). RT-PCR analysis of
RNA isolated from cultures treated solely with Shh show high expression of key
components of the pathway Ptch1, Smo and Gli1, either after 48 hours (lane 1)
or after 72 hours (lane 2) in culture. Smo and Gli1 expression are
downregulated after 24 hours of Bmp2 treatment (lane 6), whereas Ptch1
expression is not regulated by Bmp2. This is in concordance to the in vivo
situation where Smo and Gli1 expression are downregulated along cerebellum
development, whereas Ptch1 and Shh expression are maintained to adulthood
(Traiffort et al., 1999).
Experiment shown reflects observations made in three separate experiments.
Bmp2 and Bmp4 inhibit proliferation of granule cell precursors in the EGL
Our data supports the suggestion that Bmp2 and Bmp4 exert a potent
antagonistic activity on the Shh-induced proliferation of granule cell
precursors in vitro. However, cell proliferation/differentiation of cerebellar
granule neurones is also finely regulated by extracellular matrix
glycoproteins (Graus-Porta et al.,
2001; Pons et al.,
2001
) and by proteoglycans
(Rubin et al., 2002
). Thus, in
order to test the in vivo relevance of BMP activity seen in primary cultures,
we adapted an organotypic slice culture of cerebellum that maintains cell-cell
and cell-matrix interactions occurring during normal granule neuron
development. Beads soaked either in PBS (control beads), Shh (1 µg/ml),
Bmp2 (0.1 µg/ml), Bmp4 (0.1 µg/ml) or Bmp7 (0.1 µg/ml), were
implanted close to the EGL (Fig.
4A), and organotypic slices were maintained for 3 days in culture.
Four hours prior to fixation cultures were pulsed-labelled with BrdU (50
ng/ml) to label proliferating cells, fixed and co-immunostained with anti-BrdU
antibody and with an anti-calbindin antibody to label Purkinje neurones. Only
those beads that at the end of the culture period were properly placed either
above or within the Purkinje cell layer
(Fig. 4B) were further
analysed. At least eight to ten beads loaded with each purified protein were
analysed in three independent experiments. The total numbers of
BrdU-immunostained nuclei, in a fixed area surrounding the beads, were counted
on a Leica confocal microscope and quantitative data were expressed as
mean±s.e.m.
|
BMP-dependent granule neuron differentiation is mediated by Smad5
BMP proteins signal through membrane localised serine/threonine kinase
receptors, which propagate the signal by phosphorylating Smads. Smad1 or the
closely related Smad5 and Smad8 proteins are the only known BMP receptor
substrates capable of signal transduction. In order to define which Smad
protein might be transducing the BMP-mediated antagonism of Shh in granule
neuron development, vibratome sections of developing cerebella were hybridized
with probes to Smad1, Smad5 and Smad8. Smad1 expression is
restricted to the external germinal layer (EGL), from the earliest stage
analysed (chick HH38, Fig. 5A)
until late in cerebellar development (Fig.
5B-D). Smad5 however, is expressed in both the EGL and
the IGL from HH38 (Fig. 5E).
Interestingly, smad5 expression in the IGL is particularly high in a
layer just below the Purkinje cells (Fig.
5G,H), where early differentiated granule neurones reside, an area
corresponding to that of bmp2 expression
(Fig. 1D). Smad8 is
not expressed in the developing cerebellar cortex at any stage analysed
(Fig. 5I,J). Strong
smad8 expression observed in a motor nucleus
(Fig. 5K,L) indicates that the
probe and hybridisation procedure are effective and therefore confirms the
absence of expression in the cerebellar cortex. As phosphorylation of Smad
proteins is a pre-requisite for signal transduction, we immunostained
vibratome sections of mouse and chick cerebellae with an antibody against
phosphorylated-Smad1/5/8 (P-Smad1/5/8). Immunostained sections revealed the
presence of phosphorylated forms of Smad1/5/8 in nuclei that are starting to
migrate away from the EGL, in a pattern corresponding to that of bmp2
and smad5 mRNAs expression (Fig.
5M-O).
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Discussion |
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Sonic hedgehog-induced proliferation of cerebellar granule precursors is inhibited by bone morphogenetic proteins
Shh, although initially recognised for its role in embryonic patterning
(reviewed by Ingham and McMahon,
2001), has more recently been described as having a role in the
proliferation of neural precursors (reviewed by
Martí and Bovolenta,
2002
; Ruiz i Altaba, 2002a). Shh signalling is required for
expansion of neural precursor cells in the EGL of the developing cerebellum
and is the most potent known mitogen for cerebellar granule precursors in
vitro (Dahmane and Ruiz i Altaba,
1999
; Kenney and Rowitch,
2000
; Pons et al.,
2001
; Wallace,
1999
; Wechsler-Reya and Scott,
1999
). We now know that Nmyc1 is a direct target of the Shh
pathway that functions to regulate cell cycle progression in granule cell
precursors by increasing G1 cyclin expression
(Kenney and Rowitch, 2000
;
Kenney et al., 2003
).
Furthermore, mutations in several components of the Shh pathway seem to
account for most cases of desmoplastic medulloblastomas (reviewed by
Rubin and Rowitch, 2002
;
Wechsler-Reya and Scott,
2001
). Thus, the control of Sonic signalling is likely to be a key
point in the normal developmental programme of cerebellar granule
neurones.
We, and others, have previously reported that extracellular matrix
glycoproteins (Pons et al.,
2001) and FGFs (Wechsler-Reya
and Scott, 1999
; Pons et al.,
2001
) are able to differentially modulate but not to totally
suppress Shh-mediated proliferation of granule cell precursors. TGFß
signalling generally has a negative effect on cell growth such that
inactivation of this pathway contributes to tumourigenesis (reviewed by
Shi and Massagué,
2003
). Among the TGFß superfamily, BMPs have opposing
activities to hedgehogs in many developmental paradigms
(Lee and Jessell, 1999
;
Mekki-Dauriac et al., 2002
;
Patten and Placzek, 2002
;
Zhu et al., 1999
). Thus the
expression of Bmp2 and Bmp4 in proliferating and early
differentiated granule neurones of the cerebellum shown here, indicates the
possibility of a functional interaction with Shh in the regulation of granule
cell development. In primary cultures of granule cell precursors, Bmp2 and
Bmp4 are able to totally overcome Shh-induced proliferation. Furthermore, in a
pseudo-in vivo situation, such as organotypic slice cultures, Bmp2 and Bmp4
significantly reduce granule cell precursor proliferation at the EGL,
proliferation induced by endogenous Shh. These results strongly suggest that
Bmp2 and/or Bmp4 are potent inhibitors of the Shh pathway during normal
development of the cerebellum and raise the interesting point of whether these
molecules could also control hedgehog pathway activity in medulloblastoma
growth. In accordance with this, a recent publication has suggested that Bmp2
may mediate retinoid-induced apoptosis in medulloblastoma cells
(Hallahan et al., 2003
).
Bmp7, a slightly more divergent member of the BMP family, is expressed in a
different cell type population and exhibits an apparently different role.
Similar to previous reports for Shh
(Dahmane and Ruiz i Altaba,
1999; Traiffort et al.,
1999
; Wallace,
1999
; Wechsler-Reya and Scott,
1999
), Bmp7 is expressed in migrating and settled Purkinje
neurones. Bmp7 had no significant effect on the Shh-mediated proliferation of
granule cell precursors, either in primary cultures or in organotypic slice
cultures, thus leaving the role that Bmp7 might be playing in cerebellar
development unresolved.
Bmp2 activity in the developing cerebellum is mediated by Smad5
Although the diverse TGFß ligands elicit quite different cellular
responses, they all share a highly conserved signalling pathway. Ligand
binding to type I and type II receptor serine/threonine kinases at the cell
surface initiates signalling through phosphorylation of the Smad proteins.
There are eight distinct Smad proteins among which Smad1, Smad5 and Smad8 are
directly phosphorylated and activated by Bmp signalling. Phosphorylated
Smad1/5/8 undergoes homotrimerization and formation of heteromeric complexes
with Smad4. The activated Smad complexes are then translocated into the
nucleus and, in conjunction with other nuclear co-factors, regulate the
transcription of target genes
(Massagué, 2000;
Massagué et al., 2000
).
We asked which Smad protein might be transducing the Bmp-mediated antagonism
of Shh activity in the developing cerebellum. We show that Smad1 and
Smad5 are both expressed in the developing cerebellar cortex,
although in different cell populations. Whereas Smad1 expression is
restricted to the EGL, where granular cell precursors proliferate, Smad5 is
expressed in early differentiated granule neurones. We used a phospho-specific
antibody to Smad1/5/8 (anti-P-Smad1/5/8) to evaluate the activation of Smad1
and Smad5, and found that at these developmental stages anti-P-Smad1/5/8 only
labels nuclei in a pattern overlapping to that of Smad5 mRNA
expression. This expression analysis strongly suggests that the signalling
activity of Bmp2 is mediated by Smad5. Bmp4, however, which is expressed in an
overlapping pattern to that of Smad1 at the EGL, seems not to be
signalling at this developmental stages as Smad1 is not being phosphorylated.
We favour the hypothesis that expression of Bmp4 at the EGL may be
inherited from earlier developmental stages, at which Bmp4 mediates
determination of granule cell precursor
(Alder et al., 1999
), and that
later Bmp4 is apparently not active during clonal expansion and/or final
differentiation of granule neurones. However, the fact that Bmp receptor
(Bmpr) 1a and Bmpr1b are highly expressed in the EGL
(Ming et al., 2002
) suggests
that Bmp4 might be alternatively using a non-canonical Smad1/5/8 signalling
that needs to be investigated.
In primary cultures of granule cell precursors grown in the presence of
Shh, Bmp2 treatment induces strong and transient Smad5 phosphorylation.
Furthermore, we show that Smad5 overexpression is sufficient to suppress the
proliferative response to Shh and allow granule cell precursor to enter the
differentiation programme. Whether this is achieved by a direct interaction
Smad/Gli (Liu et al., 1998),
by the competition for common transcriptional co-activators
(Goodman and Smolik, 2000
) or
co-repressors (Dai et al.,
2002
; Wang et al.,
2000
) of the Smad and the Gli pathways, or by different
mechanisms, remains to be elucidated.
Shh-mediated proliferation of granule cell precursors is as well regulated
by components of the extracellular matrix
(Graus-Porta et al., 2001;
Pons et al., 2001
;
Rubin et al., 2002
). We have
previously described that the extracellular matrix glycoprotein vitronectin
stimulates CREB phosphorylation using a pathway not involving MAPK, and that
CREB signalling was sufficient to induce differentiation of granule cells.
These results revealed CREB as an essential signal for granule neuron
differentiation (Pons et al.,
2001
). We described the role of Bmp2 as a potent inhibitor of
Shh-induced proliferation, and show that the BMP pathway in the developing
cerebellum activates Smad5 phosphorylation. Whether CREB- and Smad5-mediated
transcription of target genes are two parallel pathways leading to granule
neuron differentiation, or whether there are points of crosstalk between these
two pathways remain to be determined, although a cooperation between Smads and
CREB to activate transcription in response to TGFß signalling has already
been reported in a different cell context
(Zhang and Derynck.,
2000
).
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ACKNOWLEDGMENTS |
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Footnotes |
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