File S1 - FGF/FGFR2 Signaling Regulates the Generation and Correct Positioning of Bergmann Glia Cells in the Developing Mouse Cerebellum

Table S1. Locomotor behaviors of control and Fgfr2 cKO mice. 12 weeks old male Fgfr2lox/lox (control, n = 15) and Nestin-Cre;Fgfr2lox/lox (Fgfr2 cKO, n = 12) mice were tested in the modified hole board (mHB) for horizontal and vertical locomotor abilities. Motor coordination and balance was assessed with the rotating rod apparatus (Rotarod). All values given are mean ± s.e.m. Table S2. Average proportion of Ccnd1+/Pax6− RG/BG precursors/cells among the total number of migrating Ccnd1+ and/or Pax6+ cells in each 50-µm bin in control-, FGF9- or SU5402-treated microexplant cultures. Values represent the average proportion of Ccnd1+/Pax6− RG/BG precursors/cells among the total number of migrating Ccnd1+ and/or Pax6+ cells in each 50-µm bin (distance migrated from the border of the microexplant) and for each treatment, and the 95% confidence interval estimated with a logistic model (in 8 bins and 3 treatments: total cells migrated: 1168, among them RG/BG precursors/cells: 146). Figure S1. Correlation of locomotor and cerebellar phenotypes in adult Fgfr2 cKO mice. (A) Rotarod performance (latencies to fall) of 15 control and 12 Fgfr2 cKO males. Highlighted in red are the control male with the shortest latency to fall (16 sec, ID 30064154), and two Fgfr2 cKO males with the longest (140 sec, ID 30064156) and shortest (25 sec, ID 30064164) latencies to fall. (B–G) Sagittal cerebellar sections from the adult males highlighted in red in (A), counterstained with DAPI (B,D,F) or immunostained for Calb1 (C,E,G). Note the severe cerebellar defects in the ID 30064164 Fgfr2 cKO male with the shortest latency to fall from the Rotarod (F,G). I-X, lobuli of the adult cerebellum. Scale bar (B): 500 µm. Figure S2. The ventral mid-/hindbrain region is not affected in Fgfr2 cKO mice. (A–F) Sagittal views of the ventral MHR from adult control (A,C,E) and Fgfr2 cKO mice (B,D,F), hybridized with riboprobes for Tyrosine hydroxylase (Th; the rate-limiting enzyme for dopamine and noradrenaline synthesis) (A,B), the Serotonin transporter (Sert, also known as Slc6a4, expressed on serotonergic neurons) (C,D) and the vesicular Acetylcholine transporter (VAChT, also known as Slc18a3, expressed in cholinergic neurons) (E,F). Gross anatomical alterations of these neuronal populations were not detected in the Fgfr2 cKO mice. DR, dorsal raphe nucleus; LC, locus ceruleus; LDTg, laterodorsal tegmental nucleus; RF, reticular formation (brainstem); SNc, substantia nigra pars compacta; VTA, ventral tegmental area. Scale bar (A): 500 µm. Figure S3. Fgfr expression in the developing murine mid-/hindbrain region. (A–R) Representative brightfield (A–C,G–I,M–O) and darkfield (D–F,J–L,P–R) views of the mid-/hindbrain region on cresyl-violet-stained midsagittal sections from wild-type (CD-1) mouse embryos at E14.5 (A–F; n = 5 embryos), E16.5 (G–L; n = 5 embryos), and E18.5 (M–R; n = 6 embryos), hybridized with radioactive Fgfr1 (A,D,G,J,M,P), Fgfr2 (B,E,H,K,N,Q) and Fgfr3 (C,F,I,L,O,R) riboprobes. CbA, cerebellar anlage; ChPl, choroid plexus; EGL, external granular layer; IC, inferior colliculus; PCL, Purkinje cell layer; rH, rostral hindbrain; rl, rhombic lip; Tg, tegmentum; VZ, cerebellar ventricular zone. Scale bar (C): 200 µm. Figure S4. Disruption of the anterior PCL but apparently normal RG scaffold in the E17.5 Fgfr2 cKO CbA. (A–L) Representative confocal overviews (A,B,E–L) and close-up views (C,D) of the anterior CbA on sagittal sections from control (A,C,E,G,I,K) and Fgfr2 cKO (B,D,F,H,J,L) embryos at E17.5 (n = 1 embryo/genotype), immunostained for Pax6 (cyan/green in A–D; a marker for GCPs) and Calb1 (red in A–D; a marker for PCs), or Ccnd1 (cyan/green in E–H; a marker for cycling GCPs and RG/BG precursors/cells) and Glast (red in E,F,I,J; a marker for RG/BG fibers), and counterstained with DAPI (blue in A–F,K,L; a nuclear marker). (C,D) are close-up views of the boxed areas in (A,B). (G–L) are single color channel views of (E,F), respectively. Yellow arrowheads in (D) delimit the lacking Calb1+ anterior PCL in the mutant embryos, and in (F) point at ectopically located Ccnd1+ RG/BG precursors within the mutant cerebellar VZ. White arrowheads in (F,H) delimit the distorted Ccnd1+ anterior outer EGL in the mutant embryos. EGL, external granular layer; PCL, Purkinje cell layer. Scale bars: 100 µm (A); 30 µm (C). Figure S5. SHH signaling does not appear to be affected in the CbA of Fgfr2 cKO embryos. (A–H) Representative sagittal darkfield (A,B) and brightfield (C–H) views of the CbA in E16.5 (A–D; n = 5 embryos/genotype) and E18.5 (E–H; n = 4 embryos/genotype) control (A,C,E,G) and Fgfr2 cKO (B,D,F,H) embryos, hybridized with riboprobes for Shh (A,B,E,F) and Ptch1 (C,D,G,H). Red arrowheads in (F) delimit the lacking Shh+ anterior PCL in the mutant embryos. EGL, external granular layer; PCL, Purkinje cell layer. Scale bar (A): 100 µm. (PDF)