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First published online 4 April 2007
doi: 10.1242/dev.02840
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1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66,
4058-CH Basel, Switzerland.
2 Developmental Genetics, DKBW Centre for Biomedicine, University of Basel
Medical School, Basel, Switzerland.
* Author for correspondence (e-mail: denis.monard{at}fmi.ch)
Accepted 21 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Protease nexin 1, Sonic hedgehog, Low density lipoprotein, Receptor-related protein (LRP), Proliferation, Cerebellum, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Gloor et al., 1986;
Knauer et al., 2000). After
secretion, PN-1 is complexed with its target proteases and binds to specific
members of the low density lipoprotein receptor-related protein (LRP) family
(Strickland and Ranganathan,
2003). The ligands are internalized in endosomes via
clathrin-coated pits and are degraded in lysosomes. Simultaneously, the
intracellular LRP domain signals, via coupling, to adaptor and scaffold
proteins (Schneider and Nimpf,
2003). The role of PN-1 in the adult brain has been studied
(Kvajo et al., 2004;
Luthi et al., 1997), but
possible functions during nervous system development remained elusive. During
embryogenesis and in the postnatal brain, Pn-1 is expressed
prominently in areas of high remodelling, in which cell proliferation and fate
specification are influenced by morphogens such as sonic hedgehog (SHH)
(Kury et al., 1997;
Mansuy et al., 1993). SHH is a
key regulator of vertebrate morphogenesis
(Ingham and McMahon, 2001) and
its binding to the patched homolog 1 (PTC1; also known as PTCH1 - Mouse Genome
Informatics) receptor causes the cessation of smoothened (SMO)-mediated
inhibition of signal transduction and triggers the activation of the GLI
family of transcriptional regulators (Ho
and Scott, 2002). In the developing CNS, Shh and
Pn-1 are co-expressed in the ventral part of the mesencephalon and
myelencephalon, and in the mid-hindbrain junction, otic vesicles and
cerebellum (Dahmane and Altaba,
1999; Mansuy et al.,
1993; Wallace,
1999; Wechsler-Reya and Scott,
1999).
The developing cerebellum is a good model in which to study the regulatory
pathways that coordinate cell proliferation with cell survival and
differentiation. In rodents, this cortical structure is transiently enveloped
by the external granular layer (EGL), which consists of cerebellar granular
neuron precursors (CGNPs), which proliferate from birth until postnatal day 15
(P15). SHH is considered as the main proliferative signal of CGNPs
(Dahmane and Altaba, 1999;
Kenney and Rowitch, 2000;
Wallace, 1999;
Wechsler-Reya and Scott,
1999), a role that requires the extracellular modulation of SHH by
heparan sulfates (Rubin et al.,
2002) and the binding of the chemokine SDF-1 (also known as CXCL12
- Mouse Genome Informatics) to its receptor CXCR4
(Klein et al., 2001). By
contrast, negative regulators of SHH signalling such as vitronectin
(Pons et al., 2001),
fibroblast growth factor 2 (FGF2)
(Wechsler-Reya and Scott,
1999), BMPs (Rios et al.,
2004) and PACAP (also known as ADCYAP1 - Mouse Genome Informatics)
(Nicot et al., 2002) induce
cell cycle exit and the differentiation of CGNPs.
Our results show that PN-1 modulates the signalling activity of SHH and promotes the differentiation of CGNPs and Bergmann glia. In particular, we establish that PN-1 antagonizes SHH-induced proliferation of CGNPs. In Pn-1-deficient mice, the expression of SHH targets is enhanced in the EGL and Bergmann glia, which correlates well with the delayed differentiation of CGNPs and altered maturation of Bergmann glia. In particular, the Pn-1 deficiency causes an increase in mature granular cells. We conclude that the interaction of PN-1 with SHH is important for shaping the cerebellum during its postnatal development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Sommer et al.,
1989). RAP (also known as LRPAP1 - Mouse Genome Informatics) was a
generous gift from M. Etzerodt (University of Aarhus, Aarhus, Denmark).
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) and knock-in
(Kvajo et al., 2004) mice were
backcrossed in the C57BL6/J line for 11 generations. Heterozygous matings of
Pn-1-deficient mice allowed the comparative analysis of littermates
of all genotypes. All animal experiments were performed according to the Swiss
laws governing animal experimentation and approved by the Swiss veterinary
authorities.
Primary cultures of CGNPs
CGNPs were isolated from P5-P8 mice cerebella over a Percoll gradient, as
described previously (Hatten et al.,
1988). Purified CGNPs were re-suspended in 10% horse-serum medium
and 106 cells per well were plated in 24 well plates on glass
coverslips coated with poly-L-lysine (10 or 500 µg/ml; Fluka). After
overnight recovery, cells were cultured in serum-free medium containing 1%
stripped BSA and I-1884 supplement (Sigma). Depending on the assay, the
culture medium was supplemented with either 50, 100, 200 or 3000 ng/ml SHH,
and/or 30 or 210 nM PN-1, and/or 1 µg/ml RAP for 48 hours. Bergmann glial
cells were isolated and purified as described previously
(Hatten, 1985). For
proliferation studies, cells were incubated with 10 µg/ml BrdU for 16
hours. Mixed cultures from Pn-1-deficient mice were prepared from
individually processed cerebella to compare proliferation rates among
age-matched littermates. All coverslips were processed for BrdU immunostaining
and were counterstained with Hoechst. Pictures were acquired using a Leica DMR
microscope and a SPOT-1 digital camera. Fluorescent staining was analyzed
using Image-Pro Plus (Media Cybernetics). For proliferation index
determination, the ratio of BrdU-positive cells/Hoechst-labelled cells was
calculated over ten fields per coverslip. The averages were calculated using
four independent experiments.
Beta-galactosidase staining and activity assays
Beta-galactosidase-detection and -activity assays were performed as
described previously (Kvajo et al.,
2004). CGNPs were incubated overnight in serum-free medium with or
without FGF2 (25 or 50 ng/ml) and/or SHH (3 µg/ml), lysed and processed
using the Galacto-Star kit (Applied Biosystems), and analyzed using a
microplate reader.
Immunohistochemistry and immunocytochemistry
Cells grown on coverslips were post-fixed in 4% paraformaldehyde and the
antibodies used were: anti-beta-III-tubulin (1/200, Chemicon), 4B3 monoclonal
anti-PN-1 (1/100) (Meier et al.,
1989), anti-LRP1 (1/200; provided by D. Strickland, Department of
Biochemistry, American Red Cross, MD, USA), anti-prominin 1 (1/300, Chemicon),
anti-GFAP (1/500, Sigma). P10 wild-type and Pn-1-knockout mice were
injected intraperitoneally with BrdU (Sigma) at 100 µg/g of body weight,
and brains isolated 1 hour later. Cryostat or paraffin sections (12 µm and
6 µm, respectively) were stained using: anti-PN-1 (1/100), anti-LRP1
(1/100), anti-calbindin-28K (1/200, Sigma), anti-BrdU (1/250, BD Pharmingen),
anti-GFAP (1/1000, Sigma), anti-p27 (1/100, BD Pharmingen), anti-MATH1 (also
known as anti-ATOH1; 1/100; generous gift from J. Johnson, University of Texas
Southwestern Medical Center, Dallas, TX, USA) and an ti-doublecortin (1/100,
Santa Cruz Biotechnology). The specificity of the PN-1 antiserum was
established by the absence of staining in cerebella of Pn-1-deficient
mice (data not shown). Alexa Fluor 488 or horseradish peroxidase (HRP)-coupled
antibodies included anti-mouse (1/500, Molecular Probes; 1/1000, Amersham
Bioscience) and anti-rabbit (1/500, Molecular Probes). PN-1, LRP1 and
doublecortin immunostainings were performed using a Discovery XT automated
stainer (Ventana Medical Systems) with Ventana DAB Map detection kit
(Easwaran et al., 2003).
Antigen retrieval was achieved in CC1 and CC2 buffers (Ventana). Secondary
biotinylated antibodies were: donkey anti-goat (1/200, Jackson ImmunoResearch)
and Ventana universal secondary antibody. Signals were amplified using the
AmpMap kit with TSA (Ventana). Sections from wild-type and mutant mice were
processed simultaneously. Quantification of BrdU labelling and p27-positive
cells was performed on mid-sagittal sections in the region of the
pre-culminate fissure of lobes III and IV. The average ratio of BrdU- or
p27-positive to -negative CGNPs was determined over 200 µm of EGL for each
cerebellum.
In situ hybridization
In situ hybridizations were performed using 6 µm sagittal brain sections
and Gli1, Gli3, Shh and Ptc1 RNA probes as described
previously (Michos et al.,
2004).
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Western blotting
CGNP cultures and cerebella were homogenized in NP-40 lysis buffer.
Proteins (20 µg) were subjected to 10% SDS-PAGE and transferred to a
nitrocellulose membrane (Bio-Rad Laboratories). Antibodies were: anti-cyclin
D1 (clone 72-13G; 1/1000, Santa-Cruz Biotechnology), anti-cyclin D2 (clone
34B1-3; 1/300, Santa-Cruz Biotechnology) and anti-actin (1/3000, NeoMarkers).
Secondary HRP-conjugated antibodies were anti-mouse (1/5000, Amersham
Bioscience) and anti-goat (1/2000, Jackson ImmunoResearch). Blots were
developed using enzymatic chemiluminescence (ECL) (Amersham Bioscience) and
were quantified with ImageMaster total lab v2.00.
PN-1 uptake studies
CGNPs from P8 mice were cultured for 24 hours, pre-cultured in serum-free
medium for 2 hours and supplemented with lysosome inhibitors for 1 hour, all
at 37°C (leupeptin, 10 µg/ml; pepstatin A, 20 µM). Recombinant PN-1
(60 nM) was then added with or without RAP (1 µg/ml), P960 (25 µg/ml)
and/or P965 (25 µg/ml) and incubated for 4 hours at 37°C. Cells were
washed, stained using anti-PN-1 (1/800) and Alexa Fluor 488-coupled anti-mouse
antibodies (1/500, Molecular Probes). Hoechst-counterstained cells were
analyzed using a Zeiss LSM510 confocal microscope. For quantification, five
randomly chosen pictures were taken for each sample in each experiment and
analyzed using Image-Pro Plus.
GLI activity reporter assay
NIH3T3 cells were transiently co-transfected with 6 µg of the CAT
reporter plasmid pA10CAT6GBS [containing multiple GLI-binding sites
(Dai et al., 1999); provided by
S. Ishii, RIKEN Tsukuba Institute, Ibaraki, Japan] and 1 µg of
beta-galactosidase reporter plasmid. After 24 hours, 3 µg/ml SHH and/or 30
nM PN-1 were added during 24 hours in serum-free medium supplemented with 1%
stripped BSA. After lysis, 100 µg of proteins were used to determine CAT
activity (CAT ELISA system; Roche).
Morphological quantitation of granular layers and 3-D reconstruction
The thicknesses of the EGL and internal granular layer (IGL) were
quantified on sagittal medial sections of wild-type and mutant cerebella at
P10 and in adults. The maximal width of the EGL and IGL in lobes VI (L1) and
VIII (L2) was measured and averaged over four adjacent sections using
Image-Pro Plus. Data were analyzed using a two-way ANOVA test (n=4
for P10 animals; n=5 for adults; Graphpad Prism). For 3-D
reconstructions, 6 µm sagittal paraffin sections of P10 and adult cerebella
were stained with cresyl violet at intervals of 60 µm, and digital pictures
of all serial sections were aligned and reconstructed using ImageAccess,
AutoAligner2 and Imaris 5.0.3 (Bitplane).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and the
PN-1 protein was detected using specific antibodies. At birth (P0),
beta-galactosidase activity was detected in cells of the inner mass of the
cerebellum, corresponding to migrating Purkinje and glial cells
(Fig. 1A,B). A few
Pn-1-expressing cells were also detected in the EGL overlaying the
cerebellum, possibly corresponding to precursors
(Fig. 1B, arrowheads).
Furthermore, the PN-1 protein was detected in cells of the forming Purkinje
cell layer (PCL; Fig. 1G,H, red
arrowheads), in Bergmann glial cells (Fig.
1H, black arrowheads), diffusely in the EGL
(Fig. 1H) and in the dorsal
anterior lobes (Fig. 1G,
arrow). By P2, abundant Pn-1-expressing cells were detected in the
PCL (Fig. 1C,D). The
distribution of beta-galactosidase-positive cells was graded along the
anteroposterior axis, being absent or weak in dorsal and ventral parts, but
stronger in central lobes. Each of the positive lobes displayed lower
beta-galactosidase activity in its invaginations
(Fig. 1C). By contrast, the
PN-1 protein was rather lower in central than in dorsal and ventral regions
(Fig. 1I,J). These regional
differences in PN-1 transcript and protein distributions could be due to
secretion and internalization of the PN-1 protein by target cells (see below)
and/or to additional post-transcriptional regulation. PN-1 distribution within
the PCL is rather reminiscent of those of Shh and Gli1
transcripts at equivalent stages (Corrales
et al., 2004; Lewis et al.,
2004). In the EGL, a few PN-1-positive CGNPs were still scattered
throughout the lobes (Fig. 1D,
arrowheads). By P8, the overall level of beta-galactosidase activity had
decreased (Fig. 1E), but
remained in Purkinje and Bergmann glia cell bodies
(Fig. 1F, arrowheads). PN-1
protein was detected in dorsal lobes and their invaginations
(Fig. 1K, arrows), in Bergmann
glia (Fig. 1L, arrowheads), in
the PCL and in scattered progenitors of the EGL
(Fig. 1M, red and green
arrowheads). In adults, Pn-1 expression was weak and rather
homogeneous within the PCL (data not shown). The distribution of the major
PN-1 receptor, LRP1, was rather ubiquitous, although was higher in the PCL of
the dorsal and ventral regions (similar to PN-1, for details see Fig. S1 in
the supplementary material).
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) were PN-1
positive, whereas only few neuronal cell types expressed PN-1 (<3%; data
not shown). FGF2 is known to antagonize SHH-mediated proliferation of cultured
CGNPs (Wechsler-Reya and Scott,
1999). Interestingly, Pn-1 expression is upregulated by
FGF2 in mid-/hind-brain-derived mesenchymal cells
(Kury et al., 1997). To
determine whether PN-1 could be a target of SHH and/or FGF2 signal
transduction, CGNPs from P5 reporter mice were cultured overnight in medium
supplemented with either recombinant SHH or recombinant FGF2
(Fig. 2B). Pn-1
expression levels were elevated in a dose-dependent manner upon FGF2 addition
(Fig. 2B). In particular, FGF2
induced a 30-70% increase in Pn-1-expressing astroglial cells (see
Fig. S2 in the supplementary material).
Free or complexed extracellular PN-1 binds to LRP1, which mediates its
internalization (Knauer et al.,
1997b). LRP1 was detected in the cell body and along neurites of
all CGNPs (Fig. 2C) and
receptor-associated protein (RAP; also known as LRPAP1 - Mouse Genome
Informatics)-mediated blocking of LRPs significantly inhibited PN-1
internalization into CGNPs (Fig.
2D-G). Because RAP has a high affinity for all LRPs
(Herz et al., 1991), we used
the 12 amino acid P960 peptide derived from the N-terminal region of PN-1 to
characterize the involved LRP subtype further. This peptide has been shown to
specifically interfere with LRP1-mediated PN-1 uptake
(Knauer et al., 1997a).
Indeed, P960 completely blocked the internalization of recombinant PN-1
(Fig. 2G), which indicates that
LRP1 is required for PN-1 internalization by CGNPs.
PN-1 antagonizes SHH signalling in cultured CGNPs
In order to investigate the potential effects of PN-1 on SHH-induced cell
proliferation, CGNPs were cultured for 48 hours in the presence of SHH and/or
PN-1. Proliferating cells were labelled with BrdU during the last 16 hours to
determine their proliferation rates (Fig.
3A,B). As previously reported
(Wechsler-Reya and Scott,
1999), the addition of recombinant SHH stimulated CGNP
proliferation (Fig. 3A,B,
series 1). Such SHH-induced cell proliferation was significantly antagonized
by PN-1 using SHH at 50 or 100 ng/ml (Fig.
3A,B, series 2 and 3). Higher SHH concentrations were able to
overcome the inhibitory effect of PN-1, which is indicative of its limited
modulation potential and/or saturation of the system. To determine whether the
antagonistic effect of PN-1 is mediated by receptor competition or via an
independent intracellular pathway, cells were pre-treated with SHH for 5 hours
prior to PN-1 addition (Fig.
3B). Pre-treatment with SHH did not significantly alter the
inhibitory potential of PN-1, pointing to PN-1 having antagonistic effects on
SHH signal transduction rather than on its direct competition for receptor
binding.
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). Indeed, cyclin D1 was detected at a high level
in cells treated with SHH alone, and levels were lower by treatment with SHH
and PN-1 (Fig. 3C). These
results are consistent with the observed reduction in cell proliferation
(compare Fig. 3C with
Fig. 3A,B). Interestingly,
cyclin D1 was also downregulated in cells treated with both SHH and RAP, thus
indicating a possible requirement of LRPs for SHH-mediated stimulation of cell
proliferation. The specificity of the antagonistic effects of PN-1 on SHH
signal transduction was validated further by monitoring its ability to
interfere with SHH-induced transcriptional activation. Gli1
transcription was used as a sensitive read-out of SHH pathway activity in
CGNPs cultured for 48 hours in the presence of SHH alone or with PN-1
(Fig. 3D). Whereas SHH alone
upregulated Gli1 expression, the addition of PN-1 reduced
Gli1 transcription to basal levels, as revealed by semi-quantitative
reverse transcriptase (RT)-PCR analysis
(Fig. 3D). To substantiate
these results further, NIH3T3 cells, which are routinely used to assess SHH
signal transduction (Yao et al.,
2006), were transfected with a Gli1 reporter plasmid
(Dai et al., 1999). Treatment
of transfected cells with SHH alone caused an approximately fourfold increase
in the expression of the Gli1 reporter gene within 24 hours, which
could be blocked by PN-1 (Fig.
3E). Taken together, these results indicate that PN-1 antagonizes
SHH signal transduction and/or the upregulation of the transcriptional target
Gli1. Next, the proliferation rates of mixed CGNPs/glial cells (Fig. 3F) and enriched CGNPs cultures (data not shown) isolated from wild-type and Pn-1-deficient mice were determined in response to SHH signalling. Indeed, mixed CGNP cultures from Pn-1-/- mice displayed an almost twofold-higher basal proliferation rate and a higher sensitivity to SHH-induced stimulation of proliferation than wild-type controls. Addition of PN-1 antagonized SHH-induced proliferation of both mutant and wild-type cells (Fig. 3F). In particular, the addition of 210 nM of recombinant PN-1 reduced the proliferation of Pn-1-deficient CGNP cultures to wild-type levels (Fig. 3F). The naturally occurring chemical cyclopamine (Cp), which blocks SHH signal reception, had little effect on the proliferation of wild-type cells, but reduced the proliferation of Pn-1-deficient cultures to wild-type levels (Fig. 3F). Enriched CGNP cultures from Pn-1-deficient mice showed only a 28% increase in proliferation compared with the control (data not shown). This confirms that the glial population is the main source of PN-1 (also see Fig. S2 in the supplementary material) and provides evidence that the increased proliferation rates of Pn-1-deficient CGNP cultures could be due to overactivation of SHH signal transduction. These results indicate that PN-1 acts as a negative modulator of SHH signal transduction. Indeed, PN-1 inhibited SHH-induced differentiation of Bergmann glial cells (see Fig. S3 in the supplementary material). Taken together, these results show that PN-1 can antagonize both proliferation- and differentiation-inductive properties of SHH in culture.
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;
Lee et al., 1997). Both genes
were expressed in the EGL and by Bergmann glia in wild-type mice
(Fig. 4A,C,G,H,J). In
Pn-1-/- mice, the expression of both Gli1 and
Ptc1 was increased in cells within the outer EGL (oEGL)
[Fig. 4B,D,I,K; identified as
MATH1 (also known as anti-ATOH1)-positive CGNPs: see
Fig. 5F]. In addition, higher
levels of Gli1 and Ptc1 expression were also detected in
Bergmann glia in Pn-1-deficient mice (see Fig. S4 in the
supplementary material).
Shh and Gli3 are expressed in rather complementary
patterns and have been shown to functionally antagonize one another
(Zeller, 2004). Gli3
was broadly expressed in the EGL and in the internal granular layer (IGL) of
wild-type mice (Fig. 4E,L),
whereas its expression was lower and more-restricted in
Pn-1-deficient mice (Fig.
4F,M). Interestingly, Gli3 expression was normal in the
external part of the cerebellum but significantly reduced in fissures
(Fig. 4F). The changes are not
due to altering Shh itself, because Shh was expressed at
similar levels in both wild-type and mutant mice (predominantly in Purkinje
cells; Fig. 4N,O). The results
were confirmed by semi-quantitative RT-PCR analysis, which revealed that the
expressions of Gli1 and Ptc1 were significantly increased
while that of Gli3 was reduced in the cerebellum of
Pn-1-deficient mice (see Fig. S5 in the supplementary material).
Taken together, these studies corroborate the proposal that SHH signal
transduction is potentiated in mice lacking Pn-1.
CGNP differentiation is delayed in Pn-1-deficient mice
In Pn-1-deficient mice, the overall thickness of the EGL was not
altered at P5 and P10 (data not shown). By contrast, the Pn-1
deficiency resulted in a thickening of the oEGL and thinning of the inner EGL
(iEGL; Fig. 5A-L). The bHLH
transcription factor MATH1 identifies the early progenitors of the granular
lineage (Ben Arie et al.,
1997). The number of immature MATH1-positive CGNPs increased
slightly at P5 (data not shown) and significantly at P10 in
Pn-1-deficient mice (Fig.
5A-H), and MATH1 immunoreactivity was stronger in mutant CGNPs
(Fig. 5E,F). To analyze the
postmitotic zone of the EGL, the p27 cyclin-dependent kinase inhibitor, which
accumulates in the nuclei of postmitotic CGNPs, was used to monitor
differentiation (Miyazawa et al.,
2000). In wild-type cerebella, overall p27 staining was weak in
the oEGL and became more intense as CGNPs entered the iEGL
(Fig. 5I,K). In
Pn-1-deficient mice, the decrease in iEGL thickness was noticeable at
P5 (data not shown) and obvious by P10
(Fig. 5J-L). The intensity of
p27 staining was reduced in the expanded oEGL and was restricted to a limited
region of the iEGL (Fig. 5L).
In agreement, the ratio of p27-labelled to non-labelled cells was
significantly reduced in Pn-1-deficient mice by P10
(Fig. 5P). This delay in CGNP
differentiation was further evidenced by immunodetection of doublecortin, an
early marker for neuronal differentiation
(Gleeson et al., 1999), whose
expression was decreased in Pn-1-deficient mice (see Fig. S6 in the
supplementary material). These results show that the onset of differentiation
is delayed in CGNPs of Pn-1-deficient mice. However, during the
stages analyzed, this delay did not correlate with significantly altered
proliferation rates (Fig. 5M-O
and data not shown). Interestingly, the BrdU-labelled CGNPs remained located
close to the pial surface in Pn-1-/- cerebella
(Fig. 5N), whereas the
proliferative CGNPs were spread throughout the oEGL in wild-type littermates
(Fig. 5M, arrowheads). We also
studied whether PN-1 modulates the SHH-mediated differentiation of Bergmann
glial cells in vivo. Indeed, an overall increase in the thickness of
GFAP-positive fibres was observed in Bergmann glial cells at P10 in
Pn-1-deficient mice (Fig.
5R). The Bergmann glial fibres appeared irregular and contacted
the pial surface with larger endfeet in comparison to wild-type
(Fig. 5Q). These results
indicate that the SHH-mediated effects on the proliferation of CGNPs and
differentiation of Bergmann glia cells are potentiated in
Pn-1-deficient mice. Finally, the in vivo levels of the cell cycle
regulators cyclin D1 and cyclin D2, two SHH targets
(Kenney and Rowitch, 2000),
were evaluated. Immunoblot analysis was performed on cerebellar extracts from
P10 wild-type and Pn-1-deficient mice. The levels of cyclin D1 and
cyclin D2 were increased by 33 and 68%, respectively, in mutant cerebella
(Fig. 6A,B).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Nybakken and Perrimon (Nybakken and
Perrimon, 2002) proposed that the SHH-LRP interaction may allow
the clustering of PTC1, SMO and SHH, thus causing internalization and
potentiation of the signal. However, the biological relevance of LRP in SHH
signal transduction remained unclear. Using RAP, we provide evidence that LRP
accessibility is important for SHH-mediated stimulation of CGNP proliferation.
Proteoglycans are crucial for binding to LRP and for the subsequent
internalization of ligands, such as PN-1
(Crisp et al., 2000).
Interestingly, proteoglycans are also required for SHH-induced proliferation
of progenitor cells in the postnatal developing cerebellum
(Rubin et al., 2002). Because
PN-1 is known to bind to several LRP family members
(Croy et al., 2003), it is
possible that PN-1 and SHH could share the same LRP receptors on CGNPs. In
agreement with other studies, we demonstrate that PN-1 uptake is mediated by
LRP1. Moreover, microarray analysis indicates that, in the cerebellum,
LRP1 transcripts are at least tenfold more abundant than any other
LRPs (data not shown). Thus, LRP1 seems to be the best candidate for mediating
the PN-1 and SHH interactions during CGNP proliferation. The potential
competition for LRP-binding sites may be mechanistically similar to what has
been reported for dickkopf 1 (DKK1) and the Wnt modulator Wise (also known as
SOSTDC1 - Mouse Genome Informatics), which inhibit Wnt signalling by
interacting with LRP5 and/or LRP6 (Glinka
et al., 1998). However, we cannot exclude that the interaction of
PN-1 with LRPs also interferes with other ligands sharing this co-receptor,
such as BMP4. In fact, BMP4 is present in the EGL
(Rios et al., 2004) and
interacts with LRP2, which induces its clearance
(Spoelgen et al., 2005).
The cerebella phenotype of Pn-1-deficient mice corroborates the
results obtained by the analysis of primary CGNP cultures. The Pn-1
deficiency results in increased expression of the SHH transcriptional targets
Gli1 and Ptc1 in the oEGL, indicative of enhanced SHH signal
transduction. Furthermore, the number of immature CGNPs and the initiation of
their differentiation is enhanced in mutant mice, which is detrimental to the
postmitotic iEGL. These changes in CGNP differentiation probably underlie the
enlargement of the IGL from P10 onwards. The increase in IGL thickness follows
an anteroposterior gradient: it initiates in lobe VI and affects
more-posterior lobes as differentiation progresses. It is interesting to note
that these regions correspond to the territories with the highest PN-1 protein
levels during cerebellar development. Studies using gain- and loss-of-function
approaches have established that the SHH-mediated, controlled local increase
in the proliferation of CGNPs determines the extent of cerebellar foliation
(Corrales et al., 2004;
Corrales et al., 2006;
Lewis et al., 2004). These
authors postulate that regulation of the length and intensity of SHH
signalling is crucial to lobule formation. We now identify the PN-1
extracellular serine protease inhibitor as a valuable candidate modulator.
Indeed, SHH overexpression in transgenic mice does not alter EGL thickness or
CGNP proliferation in the oEGL between P5 and P10, but the IGL is expanded
from P8 onwards (Corrales et al.,
2004; Corrales et al.,
2006). In adult transgenic mice, all cerebellar lobes exhibit a
thickened IGL as also observed in Pn-1-deficient mice. Our study
uncovers a role of PN-1 in the cellular events that are controlled by
morphogenetic SHH signalling during cerebellar development. PN-1 may restrain
SHH-mediated proliferation and thereby contribute to the regulation of the
size and shape of the cerebellum via the fine-tuning of SHH-mediated
foliation. This would explain the rather localized gross-morphological
phenotype observed in Pn-1-deficient mice. Further investigation is
necessary to understand how PN-1 may participate in the formation and
maintenance of an anteroposterior gradient of SHH signal transduction, and how
PN-1 may potentially impact the switch from proliferation to
differentiation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1745/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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