DEV02840 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. The LRP1 receptor is expressed throughout postnatal cerebellar development. LRP1 immunodetection was performed on P0 (A,B), P2 (C,D) and P8 (E,F) cerebellar sagittal sections. Although ubiquitously expressed, LRP1 seems present at higher levels in the PCL of the dorsal anterior lobes and ventral posterior lobes (grey arrows in A,C,E), and levels are also increased in the deep fissures of all lobes. Scale bars: A, 200 μm; C, 400 μm; E, 250 μm; B-D-F, 30 μm.

• Supplemental Figure 2 -

  Fig. S2. Characterisation of PN-1-producing cells in cerebellar cultures in the absence or presence of FGF2. PN-1-producing cerebellar cells (recognized by nuclear beta-galactosidase, blue) isolated from KI reporter mice at P8 were double-labelled with prominin 1 (A,B), GFAP (C), doublecortin or NeuN antibodies (not shown). Pn-1-expressing cells were enriched in the cell populations expressing these markers (D,E). (D) The fraction of prominin 1-positive and GFAP-positive cells in comparison to all Pn-1-expressing cells is shown as a percentage. This analysis reveals that the PN-1-producing cells are mostly prominin 1-positive stem cells and GFAP-positive cells. (E) The fraction of Pn-1-expressing cells among all prominin 1- and GFAP-positive cells was also determined. Pn-1 is expressed by more than 75% of all prominin 1-positive stem cells and by more than 30% of all GFAP-positive astrocytes. This last group is also most responsive to FGF2 stimulation. Scale bars in A-C: 10 μm.

• Supplemental Figure 3 -

  Fig. S3. PN-1 antagonizes SHH-induced differentiation of Bergmann glia. Purified Bergmann glia from cerebella of P8 wild-type mice were cultured for 24 hours in the presence or absence of PN-1 (30 nM) and SHH (3 μg/ml). Subsequently, the cells were stained for GFAP (A-C) and the glia fibre thickness was measured. SHH induces glia fibre thickening (B,D), which is completely antagonised by PN-1 (C,D).

• Supplemental Figure 4 -

  Fig. S4. Expression of the SHH target genes Gli1 and Ptc1 is enhanced in the Bergmann glia layer of PN-1<^b>⁻<^/b>^/<^b>⁻ mice. In situ hybridization to detect the SHH transcriptional targets Gli1 (A-D) and Ptc1 (E-H) was performed using wild-type (PN-1^+/+; A,B,E,F) and PN-1^−/− (C,D,G,H) mice at P8. (A,B) Gli1 transcripts are expressed by wild-type Bergmann glia of the PCL (blue arrowheads), but absent from the Purkinje cells (black arrowheads). (C,D) Gli1 transcription is upregulated in PN-1^−/− mice. (E,F) The Ptc1-transcript distribution is similar to that of Gli1 in the wild type. (G,H) Ptc1 expression is also increased in the Bergmann glia layer of mutant cerebella.

• Supplemental Figure 5 -

  Fig. S5. Transcription of SHH target genes in the postnatal cerebellum of Pn-1-deficient mice. The expression of Shh, Gli1, Ptc1 and Gli3 was assessed by semi-quantitative RT-PCR in cerebellar cells isolated from Pn-1^+/+ and Pn-1^−/− mice (P2, P5 and P8). Shh expression fluctuates in mutant cells, rendering the increase statistically insignificant. By contrast, expression of the SHH transcriptional targets Gli1 and Ptc1 is markedly and significantly increased in mutant cells, whereas Gli3 expression is reduced.

• Supplemental Figure 6 -

  Fig. S6. Granular differentiation is delayed in the inner EGL of Pn-1-deficient mice. Sagittal sections from wild-type (Pn-1^+/+; A) and Pn-1^−/− (B) cerebella at P10 were processed for doublecortin immunohistochemistry. Doublecortin is detected in the neurites of the postmitotic granular cells of the iEGL. The staining intensity decreases (blue arrows in A,B) and the unlabeled oEGL appears enlarged (red arrows in A,B) in the mutant cerebellum. iEGL, inner external granular layer; oEGL, outer external granular layer. Scale bar: 50 μm.

• Supplemental Figure 7 -

  Fig. S7. Regionalized increase of the IGL in the mutant cerebellum at P10. The IGL thickness of wild-type (A,B) and mutant (C,D) cerebella was examined using Hoechst-stained midsagittal sections. P10 mutant cerebella exhibit an enlarged IGL in lobe VI, whereas posterior lobes are comparable to wild-type controls. The thickness of the IGL was visualized by evaluating the highest widths of L1 and L2 in lobes VI and VIII, respectively. (B,D) Higher-magnifications of lobe VI. Scale bars: A-C, 250 μm; B-D, 50 μm.