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First published online June 14, 2004
doi: 10.1242/10.1242/dev.01188

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Bmp2 antagonizes sonic hedgehog-mediated proliferation of cerebellar granule neurones through Smad5 signalling

Iria Rios^1,*, Rubén Alvarez-Rodríguez^2,*, Elisa Martí^1, and Sebastián Pons^2,

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

View larger version (124K): [in a new window]   Fig. 1. Expression analysis of BMPs in the developing cerebellum. In situ hybridisation was performed on vibratome sections of mouse and chick cerebellae with probes against Bmp2, Bmp4, Bmp7 and sonic hedgehog (Shh). (A) Hybridisation of a postnatal day 0 (P0) mouse with a Bmp2 probe shows expression in the EGL but weak expression in the IGL. (B,C) In a chick HH42 cerebellum, Bmp2 expression is weak in the EGL and strong in the IGL. (D) Double labelling with the Purkinje cell marker calbindin revealed that Bmp2 expression is located just beneath the Purkinje cell layer. (E) Hybridisation with a Bmp4 probe at HH38, shows expression in the EGL and weak expression in the IGL. (F,G) A similar pattern of Bmp4 expression is maintained throughout cerebellar development. (H) Hybridisation with a Bmp7 probe at HH38 reveals expression in migrating Purkinje cells. (I,J) Bmp7 expression is maintained in settled Purkinje neurones at HH42. (K) Hybridisation with a Shh probe at HH38 shows expression in migrating Purkinje cells. (L,M) Shh expression is maintained in settled Purkinje neurones at HH42.

View larger version (57K): [in a new window]   Fig. 2. Effects of treatment with Shh and BMPs on the proliferation of granule cell precursors. Cerebellar granule cells were cultured in medium without additional growth factors (control), or with Shh (3 µg/ml), Bmp2 (100 ng/ml), Bmp4 (100 ng/ml) or Bmp7 (100 ng/ml). (A) After 60 hours in culture, cells were pulsed-labelled with [3H]thymidine for 12 hours, harvested and analysed for incorporation of radioactivity. BMPs had no effect on proliferation, whereas Shh induces a high rate of [3H]thymidine incorporation. (B-E) After 72 hour in culture, cells were immunostained for the neural-specific marker ßIII Tubulin (red) and the astroglial marker Gfap (green) in control cultures (B) and cultures treated with Shh (C), with Bmp2 (D) or with Bmp7 (E). Cultures treated with BMPs were not phenotypically different from control cultures. Shh-treated cultures showed the presence of big clumps of precursor cells that showed no immunoreactivity to either neural or glial marker. All cultures are counterstained with the nuclear marker DAPI (blue).

View larger version (84K): [in a new window]   Fig. 3. Modulation of Shh induced proliferation by BMPs. Cerebellar granule cells were cultured in the absence or presence of purified Shh alone (3 µg/ml), or the same concentration of Shh together with Bmp2 (100 ng/ml), Bmp4 (100 ng/ml), Bmp7 (100 ng/ml), or with the PKA activator dibutyril cAMP (DBA). (A) After 60 hours in culture, cells were pulsed labelled with [3H]thymidine for a further 12 hours, harvested and analysed for incorporation of radioactivity. Bmp2 and Bmp4 cause a strong reduction in [3H]thymidine incorporation, equal to the reduction caused by DBA. Bmp7 had no effect on the Shh-induced proliferation. (B,C) After 72 hour in culture, apoptosis was assessed in cultures using the TUNEL reaction (green) and immunostained for the neural specific marker ßIII Tubulin (red). There was no significant difference between the numbers of apoptotic cells in cultures grown with Shh alone (B) or with Shh and Bmp2 (C). (D-G) Cells were immunostained for the neural-specific marker ßIII Tubulin (red) and the astroglial marker Gfap (green), and counterstained with the nuclear marker DAPI (blue). (D) Cultures treated with Shh and Bmp2 show a phenotype similar to that of control cultures (not shown). (E) Cultures grown in the presence of Shh and Bmp7 show a similar phenotype to those grown with Shh alone (not shown). (F,G) Cultures grown in the presence of DBA, either alone (F) or together with Shh (G) show long astrocytic processes. (H,I) Cultures grown in the presence of Bmp2, either alone (H) or together with Shh (I) show early differentiated astrocytes that express GFAP, even though they have a precursor cell morphology. (J) RT-PCR analysis of RNA isolated from cultures treated with Shh for 48 hours (lane 1), with Shh for 72 hours (lane 2), with Shh for 48 hours and with Bmp2 for the indicated periods of time (lanes 3-6).

View larger version (45K): [in a new window]   Fig. 4. Modulation of granule cell precursor proliferation in the EGL by Shh and BMPs. Organotypic slice cultures of cerebellum were maintained for 72 hours and analysed for granule cell precursor proliferation by BrdU incorporation. (A) Cerebellar slices were obtained (300 µm), cultured on polycarbonate filters. Beads soaked in either PBS, Shh, Bmp2, Bmp4 or Bmp7 were implanted close to the EGL. (B) Confocal images of cultured slices that were in toto immunostained with anti-calbindin antibody (green cells) and anti-BrdU (red nuclei). White dots indicate the location of the bead. Analysis of each culture was performed by counting total numbers of BrdU-positive cells in a fixed area surrounding the beads. (C) Quantification of proliferation in the EGL of organotypic slice cultures with different beads implanted. Values are expressed as mean±s.e.m. A significant increase in the total number of BrdU-positive cells is observed in slices with Shh beads (**P<0.001 Turkey’s Test) when compared with PBS-soaked beads. A significant decrease on the total number of BrdU-positive cells is observed in slices treated with Bmp2 (**P<0.001) and Bmp4 (*P<0.01) when compared with PBS beads. Although Bmp4 seams less active in these experiments, differences between Bmp2 and Bmp4 activities have no statistical significance (Bmp2 versus Bmp4 P<0.05). Bmp7-loaded beads induced a slight but not statistically significant increase on the total number of BrdU-positive cells (P<0.05) when compared with PBS beads.

View larger version (131K): [in a new window]   Fig. 5. Expression analysis of Smads in the developing cerebellum. In situ hybridisation was performed on vibratome sections of chick cerebellae with probes to Smad1, Smad5 and Smad8. (A) Hybridisation with a Smad1 probe at HH38 reveals strong expression in the EGL. (B,C) At HH42 Smad1 expression remains restricted to the EGL. (D) Double labelling with Smad1 and the mitosis marker Phospho-Histone3 (brown nuclei) reveals Smad1 expression (blue staining) localised in the highly proliferative external EGL. (E) Hybridisation with a Smad5 probe at HH38 shows expression in the EGL and the IGL. (F,G) At HH42, Smad5 expression is weak in the EGL but strong in the IGL, particularly in cells just below the Purkinje cell layer. (H) Double labelling with Smad5 and the Purkinje cell marker calbindin (brown neurones) reveals Smad5 labelling (blue staining) in granule neurons just below the Pc layer. (I,J) Hybridisation with a Smad8 probe at HH38 (I) and HH42 (J) shows no expression in any layer of the developing cerebellum. (K,L) Expression of Smad8 in cells of a motor nucleus at the ventral medulla. (M-O) Vibratome sections of mouse P4 (M) and chick HH42 (N,O) cerebellae immunostained with the phospho-specific Smad 1/5/8 antibody show the presence of phosphorylated forms of Smad proteins mainly at the IGL, together with few nuclei leaving the iEGL (arrows), which should correspond with Smad5 mRNA expression. The location of Purkinje cell bodies is indicated by broken lines.

View larger version (45K): [in a new window]   Fig. 6. Smad5 activity in cultures of granule cell precursors. The phosphorylation status of Smad1/5/8 was evaluated by western blotting of cells cultured for 24 hours in the presence of Shh and treated with Bmp2 for different time periods (0, 5, 15 and 60 minutes). Total cell lysates from granular cell cultures were separated by SDS-PAGE and the resulting nitrocellulose membranes blotted with an antibody against phosphorylated-Smad1/5/8 (P-Smad1/5/8) (lysate). Alternatively, cultures were immunoprecipitated with the anti-Smad5 antibody, separated by SDS-PAGE and the resulting nitrocellulose membranes blotted with an antibody against phosphorylated-Smad1/5/8 (P-Smad1/5/8) (IP, Smad5). (B-E) One-day old granular cell cultures plated on laminin and treated with Shh were transfected with DNA vectors containing EGFP (B,C) or EGFP-Smad5 (D,E). Cells were immunostained for proliferation with anti-BrdU (red), with the nuclear marker DAPI (blue) and with GFP (green). White arrows in B point indicate transfected cells (green stained in C) that are BrdU labelled. White arrows in D indicate the position of transfected cells (green stained in F) that are BrdU negative. (F) The percentage of GFP-expressing cells that were differentiated cells (cells that do not incorporate BrdU) was evaluated in each transfection group. Overexpression of Smad5 decreased the percentage of proliferating cells more than threefold when compared with the control group transfected with EGFP (from 54.8±8.5% to 21.5±5.3%).

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