Article Figures & Data
Figures
- Fig. 1.
Chimeric cerebella exhibit a range of phenotypes. Hematoxylin and eosin-stained parasagittal sections through the superior cerebellar peduncle are shown from (A) an Unc5h3/+ control, (B) anUnc5h3/Unc5h3 animal, and (C–E) threeUnc5h3/Unc5h3↔ROSA26 chimeric animals (chimeras 1, 4, and 5, respectively). The primary fissure (pr) separates the anterior lobe (to theleft) from the posterior lobe (to theright). Ectopic cerebellar cells that have colonized the colliculus are to the left of thewhite arrows; black arrows indicate intracerebellar ectopias, and asterisks denote divots in the granule cell layer. Note that the phenotypes of chimeric cerebella are intermediate to wild-type andUnc5h3/Unc5h3 cerebella. Also note that the larger the extracerebellar ectopia, the more attenuated the cerebellum, particularly the anterior lobe. Scale bar, 700 μm.
- Fig. 2.
The inferior colliculus and midbrain tegmentum are differentially colonized by Unc5h3 mutant cells in the chimeric brain. Parasagittal sections through the lateral cerebellum of (A) an Unc5h3/Unc5h3mouse and (B–D) threeUnc5h3/Unc5h3↔ROSA26 chimeras (chimeras 5, 4, and 3, respectively). Ectopias in the inferior colliculus are indicated by arrows, and midbrain tegmentum ectopias are indicated by arrowheads. Note in the mutant (A) and chimera 5 (B) that there are extensive ectopias in both the inferior colliculus and midbrain tegmentum. In contrast, only the inferior colliculus ectopia is present in chimera 4 (C), whereas the midbrain tegmental ectopia predominates in chimera 3 (D). Sections are Nissl-stained. Scale bars: B–D, 250 μm; A, 400 μm.
- Fig. 3.
The Unc5h3 mutation acts in granule cells and not Purkinje cells. Parasagittal sections of the cerebellum and adjoining inferior colliculus are shown from chimera 2 (A–C) and chimera 5 (D–H). The boundary between normal cerebellum (to the right) and the extracerebellar ectopia (to theleft) is denoted by longer black arrowsin A, B, D,E, and I. Wild-type (ROSA26) cells are labeled with the blue β-gal reaction product (A–H). A section (I) from the mutant controlUnc5h3/Unc5h3;ROSA26 mouse demonstrates that all cerebellar cells (even those in ectopic positions) are β-gal positive. Purkinje cells are labeled with a brown reaction product denoting calbindin immunopositivity (A–F). White arrows designate wild-type Purkinje cells, whereas short black arrowsdesignate mutant Purkinje cells (C,F). G, H, High magnification images of extracerebellar ectopia in the colliculus from chimera 5 near (G) and distal (H) to the normal cerebellum. The Purkinje cells are denoted as above. Thin, long arrows point to the granule cells that are of wild-type origin (ROSA26 positive) in the ectopia. Note that although there are a limited numbers of wild-type granule cells in the ectopia proximal to the cerebellum, there are virtually no wild-type granule cells in the ectopia distal to the normal cerebellum. All sections were counterstained with neutral red allowing the visualization of unlabeled, mutant granule cells. Thus, although the ectopic Purkinje cells are a mixture of genotypically mutant and wild-type cells, the ectopic granule cells are mainly all of the mutant genotype. Scale bars: A, D, 600 μm;B, E, I, 150 μm;C, F, 75 μm; G, H, 24 μm.
- Fig. 4.
The Unc5h3 mutation acts intrinsic to intracerebellar granule cells for IGL boundary formation. Shown are parasagittal sections through lateral cerebella of anUnc5h3/Unc5h3 animal (A, B), chimera 5 (C, D), and chimera 2 (E, F). The posterior cerebellar region is shown. Thearrows in A, C, andE denote the region shown at higher magnification inB, D, and F, respectively. The white, double-sided arrows(B, D, and F) mark the boundary between the IGL and the white matter. Sections inA and B are stained with hematoxylin and eosin. In C–F, all cells are stained with neutral red, and wild-type (ROSA26) cells are labeled with the blue β-gal reaction product. As shown in D andF, the ectopic granule cells are virtually all ofUnc5h3/Unc5h3 origin. Scale bars:A, C, E, 400 μm;B, D, F, 50 μm.
- Fig. 5.
Both genotypically mutant and wild-type radial glial cells are found in extracerebellar ectopias. Parasagittal sections through chimera 5 (A) and chimera 1 (B) were immunostained with an anti-GFAP antibody to label glial cells and counterstained with neutral red. Wild-type cells have one or more blue dots after the β-gal reaction. In both the extracerebellar ectopia (to the left) and the normal cerebellum (to the right), GFAP-positive cells are observed that possess the morphology of radial glial cells typical of the cerebellum (A, B). InC, GFAP-positive fibers (arrows) can be traced back to a genotypically normal glial cell soma in the ectopia. Also note the presence of a cell-free divot (asterisk) in a largely wild-type folia. Scale bars: A, B, 150 μm; C, 22 μm.
- Fig. 6.
Schematic representation of the contribution of genotypically mutant (gray) and genotypically wild-type (black) granule cells (circles) and Purkinje cells (P) inUnc5h3/Unc5h3↔ROSA26 chimeric brains. Within the cerebellum itself (right of arrow), Purkinje cells and nonectopic granule cells are a mixture of both mutant and wild-type cells, whereas the granule cells within the intracerebellar ectopia are genotypically mutant. In extracerebellar ectopias (left of arrow), Purkinje cells remain a mixture of both mutant and wild-type cells, whereas granule cells are predominantly mutant.
Tables
- Table 1.
Area (in μm2) occupied by granule cells in the cerebellum proper and ectopically in the midbrain tegmentum and colliculus (extracerebellar) and within the cerebellar white matter (intracerebellar) in mutant control (Unc5h3/Unc5h3;ROSA26), chimera control (+/+↔ROSA26) and experimental chimeric (Unc5h3/Unc5h3↔ROSA26) mice
Animal Area of IGL1-a Area of extracerebellar ectopic granule cells1-b Area of intracerebellar ectopic granule cells1-c Unc5h3 mutant; ROSA26 control1-d 1.10 × 106 9.15 × 105 6.34 × 105 Control chimeras 6 2.20 × 106 None None 7 1.92 × 106 None None 8 1.98 × 106 None None Experimental chimeras 1 1.75 × 106 1.9 × 105 None 3 1.55 × 106 6.5 × 105 3.26 × 105 5 1.35 × 106 4.94 × 105 6.00 × 105 ↵F1-a Mean area of the internal granule cell layer for each animal generated from two medial sections (bins 1 and 2) as described in Materials and Methods.
↵F1-b Mean area of cells in the extracerebellar ectopia for each animal generated from sampled sections in bins 5 and 6 as described in Materials and Methods.
↵F1-c Total area occupied by the granule cells in the intracerebellar ectopias for each animal generated from all 10 of the measured sections as described in Materials and Methods.
↵F1-d Mutant control generated in crosses betweenUnc5h3 and ROSA26 mice as described in Materials and Methods.
Chimera No. Estimate of phenotype2-b Within normal cerebellum (%) In ectopias (%) Intracerebellar Extracerebellar GC-Ant2-c GC-Post2-c PC-Ant. PC-Post. PC GC 1 1.52-d 2.0 10 10 10 10 10 70 (close) 95 (far)2-e 2 2.5 2.5 10 20 35 30 50–60 90+ 3 3.0 3.5 20 40 56 55 51 90+ 4 2.5 3.0 30 40 37 45 33 90 5 4.0 4.5 45 65 33 42 34 90 GC-Ant., Granule cell-anterior; GC-Post., granule cell-posterior; PC-Ant., Purkinje cell-anterior; PC-Post., Purkinje cell-posterior.
F2-a The percentage of cells that areUnc5h3/Unc5h3.
↵F2-b Range: 1–5 with 1 = nonmutant phenotype while 5 = Unc5h3 mutant phenotype.
↵F2-c The percentage chimerism for the nonectopic granule cells is an estimate because of variations in distribution of granule cells across the cerebellum.
↵F2-d There were no obvious intracerebellar ectopias in this chimera but only hints that the border with the white matter may be disrupted in a few locations.
↵F2-e Close refers to cells that are near the edge of the normal cerebellum, and far refers to the granule cells that are more distal to the normal cerebellum.
