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First published online 4 April 2007
doi: 10.1242/dev.02840

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Protease nexin 1 and its receptor LRP modulate SHH signalling during cerebellar development

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.

View larger version (140K): [in this window] [in a new window]   Fig. 1. Distribution of Pn-1-expressing cells and the PN-1 protein during cerebellar development. PN-1 expression was monitored by beta-galactosidase detection in the developing postnatal cerebellum of PN-1 knock-in mice (A-F) and the distribution of the PN-1 protein was determined using specific antibodies (G-M). Following beta-galactosidase detection, sagittal sections were counterstained with neutral red (A-D) or anti-calbindin (E,F) to identify Purkinje cells and Bergmann glia. At early stages [P0 (A,B) and P2 (C,D)] beta-galactosidase activity is detected in Purkinje cells and in some CGNPs (arrowheads). By P8 (E,F), the overall expression is reduced and the labelling of the EGL has vanished. Beta-galactosidase activity remains in Purkinje cells and in the surrounding Bergmann glia cell bodies (F, arrowheads). At P0 (G,H), P2 (I,J) and P8 (K,M), the PN-1 protein is detected in the PCL. High levels of PN-1 protein are observed in the Bergmann glial cells (H,J,L, black arrowheads) and diffuse staining is observed in Purkinje cell bodies and dendrites (H,J,M, red arrowheads). Some PN-1 protein is also detected in postmitotic CGNPs (M, green arrowheads). The PN-1-protein distribution is graded along the anteroposterior axis. Initially (P0), it is rather prominent in the dorsal anterior lobes (G, arrow), and it then (P2) extends to the dorsal anterior and ventral posterior lobes (I, arrows). Later (P8), PN-1 protein is present in the dorsal anterior lobes (K, arrows) and in the deep fissures of the central lobes (K, asterisks). EGL, external granular layer; IGL, internal granular layer; PCL, Purkinje cell layer. Scale bars: 200 µm in A for A,C and in G for G,I; 50 µm in B for B,D,F; 250 µm in E; 30 µm in H for H,J; 350 µm in K; 60 µm in L for L,M.

View larger version (54K): [in this window] [in a new window]   Fig. 2. PN-1 is internalized via LRP1-mediated endocytosis by all cultured CGNPs. CGNPs from Pn-1 knock-in mice (P8) were cultured for 48 hours on polylysine substrate (500 µg/ml). (A) Pn-1-expressing cells (counterstained by anti-beta-III tubulin in brown) were identified by beta-galactosidase activity (arrowheads). (B) SHH and FGF2 were tested for their ability to stimulate Pn-1 expression in cultured CGNPs from Pn-1 knock-in mice (P5). FGF2 markedly increases Pn-1 expression, whereas SHH does not. (C) LRP1 immunodetection (green) indicates that this receptor is present in/on the cell body and processes of all cells. (D-G) Uptake of recombinant PN-1 is antagonized by blocking LRP1-binding sites. PN-1 immunodetection was performed on CGNPs (P8) incubated for 4 hours with control medium (D), or with medium supplemented with 60 nM PN-1 (E), with 60 nM PN-1 plus 1 µg/ml RAP (F), or with 25 µg/ml P960 or P965 (not shown). Arrowheads show PN-1-containing CGNPs. (G) Quantitation of the mean PN-1 immunolabelling per cell. Values are expressed as mean±s.e.m. (***P<0.001; Student's t-test). Scale bars: 20 µm in A for A,C; 5 µm in D for D-F.

View larger version (32K): [in this window] [in a new window]   Fig. 3. PN-1 negatively modulates SHH signal transduction in CGNPs. (A,B) CGNPs of P5 wild-type mice cerebella were cultured on polylysine (10 µg/ml) for 48 hours alone or with recombinant SHH and recombinant PN-1, and were then pulsed with BrdU for 16 hours. (A) The ratio of BrdU-positive cells/total number of CGNPs was determined and the proliferation index normalized with respect to the control value, which was set at 100% (corresponding to 1.4% BrdU-positive CGNPs). Addition of 50 or 200 ng/ml SHH stimulates BrdU incorporation (series 1). Addition of 30 nM (series 2) or 210 nM (series 3) PN-1 together with SHH significantly inhibits the stimulation of cell proliferation. Notice that the addition of PN-1 alone inhibits proliferation in comparison to the control. Error bars indicate s.e.m. (*P<0.05; **P<0.01; ***P<0.001; Student's t-test). (B) CGNPs were treated for 48 hours with SHH alone (50, 100 or 200 ng/ml; series 1), together with 30 nM PN-1 (series 2) or SHH, which was added 5 hours prior to 30 nM PN-1 (series 3). Prior addition of SHH does not significantly alter the inhibitory effect of PN-1. (C) Following 48 hours of treatment on polylysine substrate (500 µg/ml), CGNPs were lysed and cyclin D1 expression levels were determined. Notice that cyclin D1 protein levels increase in the presence of 3 µg/ml SHH. This increase is inhibited by the addition of 30 nM PN-1 or 1 µg/ml RAP. (D) SHH signal transduction was investigated by RT-PCR analysis of Gli1 expression in CGNPs cultured for 48 hours on a proliferation-permissive substrate (polylysine; 10 µg/ml) in presence of SHH (50 ng/ml) alone or with PN-1 (30 or 210 nM). The addition of SHH increases Gli1 transcription, which can also be reversed by adding PN-1. (E) The antagonistic effect of PN-1 on SHH signal transduction was further studied using co-transfection assays in NIH3T3 cells. A CAT reporter plasmid containing GLI-binding sites and a beta-galactosidase expression plasmid were co-transfected into cells, and, 24 hours later, 3 µg/ml SHH and 30 nM PN-1 were added. A CAT ELISA assay was performed on 100 µg of cell extract prepared after 48 hours. The addition of SHH stimulates the transcriptional activity more than fourfold. This effect is blocked by PN-1. PN-1 alone has no effect. Values represent the mean relative activity of the CAT reporter enzyme after adjustment for transfection efficiency and normalization with mock-transfected controls. (*P<0.05, Student's t-test). (F) Inactivation of the Pn-1 gene enhances the proliferation rates of CGNPs. Mixed cultures of P5 wild-type (grey bars) and Pn-1-/- cerebellar cells (black bars) were treated for 48 hours with 3 µg/ml SHH, 30 nM PN-1 (PN-1 30), 210 nM PN-1 (PN-1 210) or KAAD-cyclopamine 1 µg/ml (Cp). The proliferation rates of the CGNPs were determined and results normalized to wild type (Pn-1+/+ controls). The proliferation rates are expressed as percentages (100% corresponding to 2.98% of BrdU-positive CGNPs). Values shown represent mean±s.e.m. (***P<0.0001; two-ways ANOVA test). CGNPs from Pn-1-/- mice display enhanced proliferation and increased sensitivity to SHH. The proliferation rates are reduced to wild-type levels by adding a high concentration of PN-1 or by blocking SHH signal transduction with KAAD-cyclopamine.

View larger version (108K): [in this window] [in a new window]   Fig. 4. The expression of SHH target genes is altered in the cerebellum of Pn-1-deficient mice. The distribution of Gli1, Patched 1 (Ptc1), Gli3 and Shh transcripts were examined in the cerebellum of P8 Pn-1+/+ and Pn-1-/- mice by in situ hybridization. Sagittal sections are shown for each gene at low (A-F) and high magnifications (G-O). The frame in G indicates the enlargements shown in H-O. The black frames in E,F indicate the enlargements shown in L,M. (A,G,H) Gli1 is expressed in the EGL and in the Bergmann glia of wild-type mice. (B,I) Gli1 expression is increased in the EGL of mutants and numbers of expressing precursors are expanded. (C,J) Wild-type Ptc1 expression. (D,K) Pn-1 deficiency induces an increase in Ptc1 expression levels and in the number of Ptc1-expressing precursors in the EGL. (E,L) Wild-type Gli3 expression. (F,M) Gli3 expression is decreased in the deeper regions of the lobes and within the EGL of mutant mice. The green frames in E,F outline zones of equal expression in both wild type and mutant. (N) Shh is predominantly expressed in the PCL of wild-type mice, and is unchanged in mutants (O). At least three independent cerebella were analysed for all genes shown and yielded identical results. EGL, external granular layer; IGL, internal granular layer; PCL, Purkinje cell layer. Scale bars: 250 µm in A for A,F; 30 µm in G; 20 µm in H for H-O.

View larger version (133K): [in this window] [in a new window]   Fig. 5. The Pn-1 gene deficiency delays the onset of CGNP differentiation in the EGL. (A-H) Sections from P10 wild-type (A,C,E,G) and Pn-1-deficient mice (B,D,F,H) were immunostained for MATH1 (green; Hoechst, blue). In both groups, MATH1 staining is detected in the oEGL. The zone of MATH1-positive cells is enlarged in Pn-1-deficient mice. In addition, the intensity of the MATH1 staining increases at the cellular level. (I-L) Sections from P10 wild-type and Pn-1-deficient mice were immunostained for p27 (green; Hoechst, dark blue; overlap, light blue). The wild-type EGL is divided into two zones: the oEGL, with few p27-expressing cells, and the iEGL, expressing p27 at high levels (I,K). In Pn-1-deficient cerebellum, the p27-negative oEGL is approximately twice the width of the iEGL (J,L). (M,N) Sections from P10 wild-type and Pn-1-deficient mice injected with BrdU 1 hour prior to sacrifice were immunostained (BrdU, green; Hoechst, blue). Analysis of the wild-type oEGL (M) reveals regularly dispersed BrdU-positive CGNPs (arrowheads), whereas the proliferating CGNPs of Pn-1-deficient mice are closer to the external pial border (N). (O) The ratio of BrdU positive versus negative CGNPs is not significantly altered in mutants. (P) Mutant mice show a significant decrease in the fraction of p27-labelled CGNPs. *P<0.05 (Student's t-test). (Q,R) P10 cerebellar sections of Pn-1+/+ and Pn-1-/- mice immunostained for GFAP were analyzed by confocal microscopy. Pn-1-/- Bergmann glia display higher GFAP levels together with an increased thickness of and larger endfeet (R) in comparison to wild type (Q). iEGL, inner external granular layer; oEGL, outer external granular layer. Scale bars: 80 µm in B for A-D; 20 µm in F for E-H; 40 µm in I for I-J and in Q for Q,R; 15 µm in K for K-N and in inset in Q doe insets in Q,R.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Pn-1-deficient mice express higher levels of cyclin D1 and cyclin D2 in their cerebella. Cerebellum cortices of P10 Pn-1+/+ and Pn-1-/- mice were homogenized and processed for immunoblot analysis. After densitometric quantification, the protein levels were monitored relatively to beta-actin and normalized to the Pn-1+/+ value. Both cyclin D1 (A) and cyclin D2 (B) levels are increased in the mutant cerebellum. Values shown represent mean±s.e.m. *P<0.05 (Student's t-test).

View larger version (51K): [in this window] [in a new window]   Fig. 7. PN-1 deficiency results in localized cerebellar expansion. Wild-type (A,B) and mutant (C,D) adult-cerebellar midsagittal sections were stained for Ptc1 mRNA by in situ hybridization to reveal the IGL. The mutant cerebella show a global enlargement of the IGL in the zones facing the external side of the cerebellum. (B,D) Higher magnifications of lobe VI. (E) The thickness of the IGL was quantified measuring the highest widths in lobes VI and VIII (indicated as L1 and L2 in A-D). At P10, mutant cerebella exhibit a thicker IGL specifically in lobe VI (+67% compared to wild type). In the adults, this phenotype is still apparent (+60%). The thickness of lobe VIII is also increased, but to a lesser extent (+41%). IGL, internal granular layer; ML, molecular layer. (n=4 for P10; n=5 for adult; *** P<0.001; two-ways ANOVA test.) Scale bars: 300 µm in A,C; 100 µm in B,D.

View larger version (75K): [in this window] [in a new window]   Fig. 8. 3-D reconstruction of representative wild-type and Pn-1-deficient adult cerebella. (A-F) Half-cerebella of two representative wild-type (Pn-1+/+; A,C,E) and Pn-1-deficient mutant (B,D,F) mice are shown as 3-D reconstructions from different angles. (A,B) The cerebella shown as mid-sections. Notice that the overall size of the mutant cerebellum (B) is increased. In particular, lobes V and IX appear extended in the direction of the yellow arrows. (C,D) The cerebella shown from a posterior angle. In the mutant mouse, lobe IX (yellow arrow), again appears enlarged in its mid-part (red double arrows). (E,F) View from the mid-anterior side. The central part of the mutant cerebellum appears clearly enlarged (red double arrows) and lobe IX (yellow arrows) protrudes more than in its wild-type counterpart.