Fig 2: En1/2 are required for the survival of the medial eCN and maintenance of PC numbers.(A–F) Immunofluorescent analysis and quantification of the cerebellar area using DAPI at E15.5 (A,B) and E17.5 (C,D) showing a significant reduction in area at E17.5 (F; Student’s t-test) but not at E15.5 (E; Student’s t-test) in Atoh1-En1/2 CKOs compared to littermate controls (n = 3/condition). A slight delay in foliation was also observed at E17.5 (* base of folds). (G–N) Immunofluorescent analysis of TDTom and TUNEL on sagittal sections from a Atoh1-TDTom (G–H, K–L) and Atoh1-TDTom-En1/2 CKO (I–J, M–N) embryo showing that apoptosis is increased in the precursors of the medial and intermediate eCN of mutants at E15.5 (I–J) and at E17.5 (G–L). (O–R) Representative images of sagittal sections at the level of the intermediate CN in (S,T) stained for MEIS2 and TDTom in Atoh1-TDTom (O,P) and Atoh-TDTom-En1/2 CKOs (Q,R). (S,T) Quantification of the average number of MEIS2+ eCN cells in every second sagittal section along the medial-lateral axis at E15.5 and E17.5 in Atoh1-TDTom compared to Atoh-TDTom-En1/2 CKOs (n = 3 for each genotype) showing reduction in the medial/intermediate eCN at E17.5. eCN counts are plotted as bins of 5% percent of the mediolateral distance (E15.5: Two-way ANOVA F(1,42)=2.182, p=0.1471, E17.5: Two-way ANOVA, F(1,42)=23.67, p<0.0001). (U,V) Quantification of PCs (FOXP2+ cells, see A-D) showing reduced PC numbers in Atoh1-En1/2 CKOs compared to controls (n = 3 each genotype) at E17.5 (V) but not at E15.5 (U) (E15.5: Student’s t-test, p=0.88). Scale bars: 100 µm.

Fig 3: Neurogenic timing-dependent H2B reporter labeling of IO neurons. (a–p) Coronal sections of the IO at approximately 35% (left panel) and 65% (right panel) levels from the caudal edge in eight G2A::H2B mice with tamoxifen injection at different embryonic stages (TM9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, and 13.0). Signals indicate immunostaining for FoxP2 (red) and H2B reporter labeling (cyan). Subdivisions of the IO are indicated by white lines. Filled and open arrowheads indicate some reporter-labeled neurons with and without FoxP2 labeling, respectively. (q, r) Definition of IO subdivisions shown in drawings of left IO in coronal sections at the two levels. “i”, “l” and “m” indicate the intermediate, lateral and medial subdivisions of some IO subnuclei. (s) Higher magnification of the IO in G2A::H2B mouse with TM10.5. H2B reporter labeling in IO neurons was co-localized with the FoxP2 labeling (white circles). On the contrary, H2B reporter labeling outside the IO was not co-localized with the FoxP2 signal (white squares). The imaged area of (s) is shown in Supplementary Fig. 2 with a square. Created with Adobe Illustrator-10.3, Adobe Photoshop-7.0, and Zen 2.6. See the separate list for abbreviations.

Fig 4: Purkinje cell clusters in coronal sections of the left E14.5 mouse cerebellum identified by marker expression profiles. A–E, Sections at different caudorostral levels indicated by percentile. Fluorescent signals of immunostaining for Corl 2 (red), Pcdh10 (blue) and FoxP2 (green) are merged. White dashed lines indicate the boundary of recognized clusters. Inset in each panel shows drawings of recognized clusters at half magnification. Squares indicate areas that are magnified. F–H, Magnified images showing PC-free gaps (arrowheads) between some clusters. I, Dorsal view of the 3D reconstruction of clusters. Red lines indicate the rostrocaudal level of the section in each panel. Scale bar in E (100 µm) applies to A–E. Scale bar in F (100 µm) applies to F–H. Abbreviations, c-l, c-m, d, dl, l, m, ml, rdl, vl, names of E14.5 clusters; C, caudal; D, dorsal; DCN, deep cerebellar nucleus; L, lateral; M, medial; R, rostral.V, ventral.

Fig 5: Purkinje cell number predicts folding amount.A-D) Sagittal midline sections of L6–7 stained with FoxP2. Scale bar: 100µm E) Purkinje Cell density. F) Purkinje cell are closely packed in FVB/NJ at the start of the critical period. G, H) The percentage of Purkinje cells within L4–5, L6–7, and L8. Scale bar: 300µm I). L6–7 has a reduced number of Purkinje cells in C57Bl/6J. J-L) Both strains have similar numbers, densities, and distributions of Purkinje cells at E16.5, C57Bl/6J: n = 4, FVB/NJ: n = 3, M,N) Sagittal midline sections stained with P27, EDU, and DAPI. Solid line: exposed surface of lobule. Scale bar: 100µm O). The proliferation rate is unchanged between strains. P) The relative size of the proliferating portion of the EGL is unchanged. (mean, s.d.)