Analysis of Cerebellar Development in math1 Null Embryos and Chimeras

Normal cell proliferation and absence of cell death in the E15.5 math1^{β-Gal/β-Gal} rhombic lip. Sagittal view of E15.5 math1^{β-Gal/β-Gal} (A, C, E, F) and E15.5 +/+ (B, D) rhombic lip, with the ventricular zone (VZ) at the top left of each image. A, B, Cresyl violet-stained sections demonstrating the presence of mitotic figures (white arrows) in both the math1^{β-Gal/β-Gal} (A) and +/+ (B) rhombic lip. In B, the arrow also demarcates the interior face (left) and the exterior face (right) of the rhombic lip. In C and D, BrdU was injected at E12.5. The presence of BrdU-positive cells (black arrows) confirms that there is active proliferation in both the math1^{β-Gal/β-Gal} (C) and +/+ (D) rhombic lip. E, F, TUNEL assay for apoptotic cell death reveals no cell death in the math1^{β-Gal/β-Gal} rhombic lip (E) compared with the TUNEL-positive cells (arrowheads) in the DNase-treated positive control (F). Scale bar, 30 μm.

Math1/lacZ-positive cells in the early embryonic math1^{β-Gal/β-Gal} cerebellum. Sagittal view of E10.5 (A) and E13.5 (B, C) math1^{β-Gal/β-Gal} cerebellum reacted for β-gal activity. A, At E10.5, Math1/lacZ-positive blue cells (arrows) are seen throughout the rhombic lip (RL). B, At E13.5, Math1/lacZ-positive blue cells are seen within the rhombic lip (RL) and migrating from the RL in a rostral direction in the superficial cerebellum (arrows). In C, Math1/lacZ-positive blue cells (arrows) appear to be migrating inward from the migratory stream in the superficial aspect of the cerebellum. Scale bar: A, C, 50 μm; B, 157.5 μm.

math1^{β-Gal/β-Gal} ↔ +/+ chimeras demonstrate an abnormal EGL with associated abnormalities in foliation. Sagittal view of P0 +/+ (A) and math1^{β-Gal/β-Gal} <-> +/+ (B, C) lateral cerebella and P5 +/+ (D) and math1^{β-Gal/β-Gal} <->+/+ (E, F) medial cerebella stained with cresyl violet. B, C, E, F, In all chimeric cerebella, there are regions of the EGL that are severely disrupted or absent (arrows and arrowheads). Where there are gaps in the EGL, foliation is disrupted compared with wild-type cerebella in A and D. Where there are large gaps in the EGL, as seen in B (arrows), foliation is absent. In some cases, where the EGL is present near the base of a folia (B, E, arrowheads), there is hyperextension of that folia (asterisk). The dark lining of the cerebellum in B and C, which has acellular areas opposite the arrows, is attributable to India ink used to highlight the gross cerebellar abnormalities in the unsectioned brain. Scale bar: A-C, 150 μm; D-F, 300 μm.

Three-dimensional reconstructions of math1^{β-Gal/β-Gal} ↔ +/+ chimeric cerebella. In all of the reconstructions, EGLs containing granule cells are gray, whereas areas without granule cells in the EGL are white. A, In this P0 chimera, the agranular EGL traverses several lobules in the posterior cerebellum, beginning at the midline and ending in the initial segments of the hemisphere. B, In this P5 chimera, the agranular zone of the EGL is located to a circumspect area starting medially in lobule VIII and progressing to the interface of the vermis and hemisphere. Lobule VIII of this brain is abnormally extended in the posterior direction (outlined in white dashed line), and this extension starts at the point that the agranular EGL starts. C, In this P5 chimera, the agranular region is located in the posterior cerebellum, extending laterally along the superior surface of lobule 6. The lobule containing the agranular EGL (white arrow) is shorter than the same lobule in control brains. D, In this P5 cerebellum, three distinct areas of the EGL are agranular. The largest gap (g1) traverses the entire medial to lateral extent of the cerebellum, without gaps. In the medial cerebellum, the agranular area encompasses much of the posterior cerebellum, whereas in the hemisphere the agranular region is more discrete. Two smaller agranular regions were seen in this brain that were oriented in the anterior-to-posterior axis. A, Anterior; P, posterior; M, medial; L, lateral.

math1^{β-Gal/β-Gal} ↔ +/+ chimeras demonstrate severe foliation defects in the P12 and P21 cerebellum. Sagittal view of P12 +/+ (A) and math1^{β-Gal/β-Gal} ↔ +/+ (B, C) medial cerebella and P21 +/+ (D) and math1^{β-Gal/β-Gal} ↔ +/+ (E) lateral cerebella, and coronal view of P21 math1^{β-Gal/β-Gal} ↔ +/+ cerebellum (F) stained with cresyl violet. In all chimeric animals (B, C, E, F), foliation is disrupted compared with wild-type cerebella in A and D. In C, the absence of foliation is associated with the almost complete lack of an IGL. In B and E, folia (arrowheads) appear to have collapsed inward, and in F there is hyperfoliation. In all cases, there is a reduction in the size of the cerebellum compared with the wild type (insets: compare B, C with A; compare E with D). Scale bar: A, D, F, insets, 600 μm; B, C, E, 300 μm.

Purkinje cell lamination defects are present in regions where granule cells are deficient in the math1^{β-Gal/β-Gal} ↔ +/+ cerebellum. Coronal view of a P12 +/+ (A) and math1^{β-Gal/β-Gal} ↔ +/+ (B) cerebellum and sagittal view of a P21 +/+ (C) and math1^{β-Gal/β-Gal} ↔ +/+ (D, E) cerebellum immunostained for the Purkinje cell marker calbindin and counterstained with cresyl violet. In the P12 (A) and P21 (C) +/+ cerebellum, Purkinje cells (arrowheads) are aligned in a single layer between the molecular layer (ML) and the IGL. In all chimeras (B, D, E), the Purkinje cell layer is disrupted where the granule cell layer is disrupted. Where the granule cell layer is absent, Purkinje cells (asterisk) are in unorganized clusters (D, inset). In adjacent regions where the granule cell layer is intact, Purkinje cells (arrowhead) are aligned in a single layer. When the granule cell layer is disrupted in the anterior cerebellum (B, E), Purkinje cells (arrows) migrate into the inferior colliculus (ic). Scale bar: A-E, 400 μm; insets in D, E, 30 μm; inset in B, 15 μm.

math1^{β-Gal/β-Gal} granule cell precursors are not generated in the math1^{β-Gal/β-Gal} ↔ +/+ cerebellum. Coronal view of a P0 math1^{β-Gal/β-Gal} ↔ +/+ (A, B) and math1^β-Gal/+ ↔ +/+ (C, D) cerebellum and P12 math1^{β-Gal/β-Gal} ↔ +/+ cerebellum (E) reacted for β-gal activity and counterstained with neutral red. In the high percentage math1^{β-Gal/β-Gal} cerebellum in A and B, there is no discernable EGL present compared with the EGL in a high percentage math1β^-Gal/+ chimeric cerebellum in C and D. However, math1^{β-Gal/β-Gal} blue cells (arrows) are seen in the medullary vellum and choroids plexus in A and deep to and within the PCP in B. Math1/lacZ-positive blue cells are found in the same location in the math1^β-Gal/+ ↔ +/+ cerebellum in D. In E, the P12 math1^{β-Gal/β-Gal} ↔ +/+ cerebellum, math1^{β-Gal/β-Gal} blue cells (arrowheads) are found within the molecular layer (ML) and adjacent to Purkinje cells. In the IGL in E, large math1^{β-Gal/β-Gal} blue cells (arrows) resembling Golgi type II cells are found among the much smaller +/+ red granule cells. Scale bar: E, 15 μm; B, D, 30 μm; A, C, 180 μm.

Some Math1/LacZ-positive cells in the P12 math1^{β-Gal/β-Gal} ↔ +/+ cerebellum are also GFAP positive. A-D, Sagittal view of the agranular P12 math1^{β-Gal/β-Gal} ↔ +/+ cerebellum in Figure 10B, immunostained for the neuronal marker NeuN (A, arrowheads), the glial marker GFAP (B, arrow), and the Math1/LacZ+ marker β-gal (C, arrow). In the merged image (D), most of the β-gal-immunopositive cells were also positive for GFAP. Scale bar, 30 μm.