Figure 2. Null and Hypomorphic Foxc1 mutations caused posterior cerebellar foliation defects due to mismigration of cells destined to form the posterior vermis.

(A–J) Lineage analysis of the Lmx1a-cre+ cells in the wild-type mice showed tdTomato expression limited to the RL, EGL and presumptive IGL. Postnatally, fate-mapped cells populated the posterior vermis but did not abut the 2^o fissure (C, white arrows). In the wild-type embryonic cerebellum, these cells were present underneath the EGL directly underneath the pial surface (A, E, G, I; white arrow). In Foxc1^hith/hith Lmx1a-cre tdTomato mice, cells migrated out of the RL in multiple ectopic streams (B, yellow arrows). Postnatally, in the Foxc1^hith/hith mutant cerebellum, ectopic tdTomato+ cells were present along the ventricular surface and the inner cerebellar core (D, yellow arrows). In Foxc1^-/- mice (F,H,J), aberrantly migrating Lmx1a-cre tdTomato+ cells were evident by e14.5 in the core (F) and found in the VZ by e15.5 (H, yellow arrow), with an extensive VZ surface presence by e17.5 (J; yellow arrow). Additionally, at e17.5, a large number of fate-mapped mutant cells were abnormally retained in an enlarged RL (J). None of the mutant internal tdTomato+ cells were Sox9+ (K,k), Skor2+ (L,l) or Pax2+ (M,m), and thus had not undergone a VZ lineage fate-switch. A subset of the fate-mapped cells were Tbr2+ (N,n, arrows), as expected of RL-derived unipolar brush cells. All tdTomato+ cells were Pax6+ (O–P). This indicated that they retained their RL origin despite aberrant migration. A subset of the Pax6+ cells is Ki67+ (O, o, Q; yellow arrows) indicating that they retain their ability to divide, while some tdTomato+ cells in the RL (asterisk) are β-III Tubulin+ (R) and Ki67- indicating that they may have differentiated precociously. Scale Bar = 100 µm (A–D, K–Q), 50 µm (E–J).

DOI: http://dx.doi.org/10.7554/eLife.20898.003

Figure 2—figure supplement 1. A significant number of ectopic tdTomato+ cells are found in the Foxc1 mutant cerebellum.

(A) Quantification of Lmx1a+ cells in the RL of WT and Foxc1 null mutants indicate that there is no difference in Lmx1a expression. p=0.17. Scale bar = 20 µm (B) Quantification of tdTomato+ cells present outside the EGL and RL area in WT and Foxc1 null mutants indicate that there is a significantly higher number of tdtomato+ cells present in the mutant, many of which are ectopic in nature. ***p<0.005 (C) Quantification of Ki67+ cells in the VZ of WT and Foxc1^hith/hith mutants indicate that there is no difference in proliferation. p=0.4. Scale bar = 100 µm (D) Mid-hindbrain expression of Foxc1 targets in e12.5 wild-type (black) and Foxc1^hith/hith (grey) littermate embryos, assayed by qRT-PCR. Foxc1 reduction decreases hindbrain mesenchyme expressed genes (Tgfb1, SDF1α, Bmp2 and Bmp4), but not neural tube expressed genes (Fgf15 and Cxcr4). *p<0.05, **p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.20898.004

Figure 2—figure supplement 2. Ectopic populations of granule cell progenitors and Purkinje cells are found in both the Foxc1^-/- and Foxc1^hith/hith mutants.

(A–H) Sagittal sections of the e19.5 wild-type (A,E,G) and Foxc1^-/- (B–D, F,H) cerebellum stained with Cresyl violet (A,B), Ki67 (C) and Pax6 (A; inset and D), showed the presence of ectopic GCPs in the Foxc1^-/- cerebellum migrating precociously into the IGL from the pial surface (C,D; arrows). Multiple ectopic Purkinje cells were also found in the Foxc1^-/- cerebellum (F,H, arrows, yellow box) as indicated by Calbindin (E,F) and Foxp2 staining (G,H). In P16 wild-type mice, Calbindin staining (I–L) demarked a monolayer of Purkinje cells (I). In P16 and P60 Foxc1^hith/hith mice, Purkinje cells were disorganized, arranged in multiple layers (J,K; arrows), and ectopically embedded in the IGL (L, white box). Sagittal sections of the WT (M,O) and Foxc1^hith/hith cerebellum (N,P) stained for GFAP (M,N) and Laminin (O,P) indicated that Bergmann glial fibers and the pial surface were structurally normal in the Foxc1^hith/hith mutant cerebellum. Scale Bar = 100 µm.

DOI: http://dx.doi.org/10.7554/eLife.20898.005