Figure 1.
RERE is expressed in the developing cerebellum.
A–D. Mid-sagittal sections of the vermis region were prepared from wild-type embryonic and postnatal mouse cerebellums at E15.5 (A), E17.5 (B), P0 (C), and P14 (D) and were stained with an anti-RERE antibody. RERE-positive cells were labeled with a red color and DAPI was used for counter staining. Inserts represent high magnification views of the boxed areas. A. RERE-positive cells are detected in the cortex of the cerebellum but not in the rhombic lip at E15.5. B. RERE expression is maintained in the entire cerebellum at E17.5. C–D. RERE immunoreactivity is particularly strong in the Purkinje cell layer at P0 (C) and P14 (D). EGL, external granule cell layer; IGL, internal granule cell layer; Mb, midbrain; ML, molecular layer; PCL, Purkinje cell layer; RL, rhombi lip. Scale bar = 100 µm.
Figure 2.
RERE is not expressed in granule cell precursors (GPCs) but is expressed in Purkinje cells.
A–C. Mid-sagittal sections of the vermis region were prepared from wild-type mouse cerebellums at E18.5 (A), P14 (B) and P7 (C). A. Mid-sagittal sections of the vermis were stained with anti-RERE antibody (red color) and anti-NR2F2 antibody (green color). DAPI was used for counter staining. A subset of Purkinje cells labeled with NR2F2 is positive for RERE at E18.5. B. Purkinje cells labeled with mouse monoclonal anti-calbindin antibodies (green color) were also stained with anti-RERE antibodies at P14. RERE expressing cells were also identified in the molecular layer and in the internal granule cell layer at P14. C. RERE immunoreactivity was not detected in PAX6-positive cells in the vermis regions of wild-type cerebellums at P7. EGL, external granule cell layer; IGL, internal granule cell layer; ML, molecular layer; PCL, Purkinje cell layer. Scale bar = 200 µm.
Figure 3.
Foliation of the principal fissures is delayed in the cerebellums of Rereom/eyes3 embryos.
A–F. Mid-sagittal sections of the vermis were prepared from cerebellums of wild-type and Rereom/eyes3 embryos and mice at E18.5 (A–B), P0 (B–C) and P3 (E–F). The Purkinje cell layer was marked using a rabbit polyclonal anti-calbindin antibody. Asterisks indicate the base of each principal fissure. A–B. At E18.5, all of the principal fissure were formed in the cerebellum of wild-type embryos (A) but the preculminate fissure, the primary fissure and the posterolateral fissure were barely appreciable in the cerebellums of Rereom/eyes3 embryos (B). The secondary fissure was not seen in the cerebellums of Rereom/eyes3 embryos (B). Arrows mark each principal fissure. C–D. Delayed development of principal fissures was restored in the cerebellums of Rereom/eyes3 embryos at P0 (D). However, the precentral fissure was found in wild-type cerebellums (arrow head, C) but was not seen in the cerebellums of Rereom/eyes3 mice (D) at P0. E–F. At P3, invagination of the precentral fissure (arrow head, E) demarcates the division of lobule III from lobule 1/II in the cerebellums of wild-type mice but the precentral fissure was not visible in the cerebellums of Rereom/eyes3 mice (F). pc, preculminate fissure; pl, posterolateral fissure; pr, primary fissure; sec, secondary fissure. Scale bar = 100 µm.
Figure 4.
Granule cell precursor proliferation was decreased in the cerebellums of Rereom/eyes3 embryos at E18.5.
A-F. Proliferation assays were performed on cerebellar vermis sections prepared from the cerebellums of wild-type and Rereom/eyes3 embryos and mice at E18.5 (A–B), P0 (C–D) and P3 (E–F) using an anti-Phospho Histone H3 antibody. A-B. The number of mitotic granule cell precursors in Rereom/eyes3 embryos appeared to be lower than that of their wild-type littermates at E18.5. C-F. Mitotic activity of GCPs was comparable between Rereom/eyes3 embryo and wild-type littermate at P0 and P3. G. Phospho-Histone H3-positive cells in the entire external granule cell layer (EGL) were counted and normalized by the area of the EGL (n≥3 with twenty slides containing three sections for each genotype). The number of Phospho-Histone H3-positive GCPs per µm2 was reduced in Rereom/eyes3 embryos at E18.5 when compared to wild-type littermates (* = p<0.03) but was indistinguishable between Rereom/eyes3 embryos and wild-type littermates at P0 and P3. ABL, anterobasal lobe; ADL, anterodorsal lobe. Scale bar = 200 µm.
Figure 5.
The level of SHH expression was reduced in the cerebellums of Rereom/eyes3 embryos at E18.5.
A. Protein extracts prepared from the cerebellums of Rereom/eyes3 embryos and wild-type littermates at E18.5 were subjected to western blot analysis using an anti-SHH antibody and an anti-HSP70 antibody. Expression of SHH in the cerebellum of Rereom/eyes3 embryos was lower than that of their wild-type littermates. Expression of HSP70 was comparable between Rereom/eyes3 embryos and their wild-type littermates. B. To quantify the level of SHH expression, the density of the SHH band was normalized by the density of HSP 70 band in same blot. The expression level of SHH was significantly decreased in the cerebellums of Rereom/eyes3 embryos when compared to the level of SHH in the cerebellums of their wild-type littermates at E18.5 (three independent experiments were performed for quantification; * = p<0.01).
Figure 6.
The migration and maturation of Purkinje cells was altered in Rereom/eyes3 embryos at E18.5.
A–D. Mid-sagittal sections of the vermis were probed with rabbit polyclonal anti-calbindin antibodies (A and B) or anti-NR2F2 antibodies (C and D). A–B. Number of calbindin-positive cells appeared to be lower in the cerebellums of Rereom/eyes3 embryos (B) when compared with those of their wild-type littermates (A). C–D. In the cerebellums of wild-type embryos, most of the NR2F2-positive cells were located in the Purkinje cell layer underneath the external granule cell layer at E18.5 (C). In contrast, the number of NR2F2-positve cells was increased in the center of cerebellar cortex of Rereom/eyes3 embryos at same time point (D). E-F. Calbindin-positive cells (E) and NR2F2-positive cells (F) were counted in the cerebellums of 18.5 embryos of both genotypes and normalized by the area of the cerebellum. The number of calbindin-positive cells/µm2 was significantly lower in the cerebellums of Rereom/eyes3 embryos when compared with wild-type littermates (* = p<0.03). However, number of NR2F2-positive cells/µm2 was comparable between Rereom/eyes3 embryos and their wild-type littermates. Quantification was performed using fifteen slides containing at least three sections from three independent littermates. pc, preculminate fissure; pr, primary fissure; PCL, Purkinje cell layer. Scale bar = 100 µm.
Figure 7.
Rereom/eyes3 mice have cerebellar foliation defects at P21.
A–B. The cerebellar vermis and hemispheres were evident in wild-type and Rereom/eyes3 mice. C–D. Mid-sagittal sections of the vermis region were stained with cresyl violet. In the cerebellums of Rereom/eyes3 mice, the precentral fissure was severely attenuated and failed to divide lobule I/II from lobule III. Red asterisks indicate the location of the precentral fissure in wild-type mice (C) and marks the abnormal lobule in Rereom/eyes3 mice (D). E–F. Sagittal sections of the cerebellar hemispheres were stained with cresyl violet. The intercrural fissure was fully developed in wild-type mice (E), but was severely attenuated in the cerebellums of Rereom/eyes3 mice (F). The black asterisks indicate the intercrural fissure. Roman letters mark corresponding lobules in the vermis of the cerebellum. CI, lobule crus I; CII, lobule crus II; PML, paramedian lobe; S, simple lobule. Scale bar = 500 µm.
Figure 8.
Branching of Purkinje cell dendrites was reduced in the cerebellums of Rereom/eyes3 mice.
A–D. Sagittal sections of the vermis of Rereom/eyes3 mice and their wild-type littermates were probed with rabbit polyclonal anti-calbindin antibodies at P14 (A, B) and P21 (C, D). A–D. A monolayer of Purkinje cells was found in the cerebellums of mice of both genotypes. Arrows point to individual Purkinje cells. Red brackets mark the dendrites of the Purkinje cells at P14. The Purkinje cell dendrites branching of Rereom/eyes3 mice (B, D) appeared to be decreased when compared that of their wild-type littermates (A, C). E. Quantification of Purkinje cells branches revealed that the Purkinje cells of Rereom/eyes3 mice at both P14 and P21 have significantly fewer branches compared to the Purkinje cells of wild-type mice at the same time points (* = p<0.03). Purkinje cells containing at least two secondary dendrites were exclusively used for quantification of dendritic branching. Quantification was performed using ten slides containing at least three sections from three independent littermates. IGL, internal granule cell layer. Scale bar = 50 µm.