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).