Migration defects of cdk5(-/-) neurons in the developing cerebellum is cell autonomous

Abstract

Cyclin-dependent kinase 5 (Cdk5) is a member of the family of cell cycle-related kinases. Previous neuropathological analysis of cdk5(-/-) mice showed significant changes in CNS development in regions from cerebral cortex to brainstem. Among the defects in these animals, a disruption of the normal pattern of cell migrations in cerebellum was particularly apparent, including a pronounced abnormality in the location of cerebellar Purkinje cells. Complete analysis of this brain region is hampered in the mutant because most of cerebellar morphogenesis occurs after birth and the cdk5(-/-) mice die in the perinatal period. To overcome this disadvantage, we have generated chimeric mice by injection of cdk5(-/-) embryonic stem cells into host blastocysts. Analysis of the cerebellum from the resulting cdk5(-/-) left arrow over right arrow cdk5(+/+) chimeric mice shows that the abnormal location of the mutant Purkinje cells is a cell-autonomous defect. In addition, significant numbers of granule cells remain located in the molecular layer, suggesting a failure to complete migration from the external to the internal granule cell layer. In contrast to the Purkinje and granule cell populations, all three of the deep cerebellar nuclear cell groupings form correctly and are composed of cells of both mutant and wild-type genotypes. Despite similarities of the cdk5(-/-) phenotype to that reported in reeler and mdab-1(-/-) (scrambler/yotari) mutant brains, reelin and disabled-1 mRNA were found to be normal in cdk5(-/-) brain. Together, the data further support the hypothesis that Cdk5 activity is required for specific components of neuronal migration that are differentially required by different neuronal cell types and by even a single neuronal cell type at different developmental stages.

Fig. 1.

Calbindin staining of sagittal sections from E18.5 cerebellum reveals migration deficiencies of Purkinje cells incdk5^−/− mutants. Anti-calbindin antibody is visualized through the red-brown DAB reaction product, seen beneath the external granule cell layer (arrowheads) in normal (cdk5^+/−) cerebellum (A). Note that the Purkinje cells in this mouse are found in their proper cortical location. Incdk5^−/− mice (B), calbindin-positive Purkinje cells are ectopically located deep in the cerebellum, near the ventricle at the bottom the cerebellum. Note the presence of a normal appearing external granule cell layer (arrowheads) in thecdk5^−/− cerebellum. Scale bar, 100 μm.

Fig. 2.

Sagittal cresyl violet-stained sections of adult wild-type (A, C) andcdk5^−/− chimeric (B,D, E) cerebellum. The chimeric cerebellum (B) is normal in foliation and cellular distribution but is reduced in size compared with wild-type cerebellum (A). The molecular layer (ml) of normal mice is cell-sparse (C, higher magnification of the boxed area in A). A monolayer of Purkinje cells is found between the molecular layer and the cell dense internal granule cell layer (igl). Beneath the internal granule cell layer are the white matter tracts, which contain myelinated axons of Purkinje cells that will synapse on neurons of the deep cerebellar nuclei. Cell density in the molecular layer of chimeras varies from region to region (D, E, higher magnification of the boxed areas in B). Despite this, the Purkinje cell layer and internal granule cell layer are normal in appearance. Large cells are seen in aberrant locations (arrowheads in the white matter of E). Scale bars: (in A) A, B, 200 μm; (in C) C–E, 50 μm.

Fig. 3.

Horizontal sections through deep nuclear region of adult wild-type (A, C, D) and chimeric (B, E–H) cerebella. Sections were double immunostained for calbindin (A–C,E, G) and Cdk5 (D,F, H). Wild-type cerebellum (A) with the ventricle (v) to the left shows prominent calbindin staining in the Purkinje cell layer of the cerebellar cortex. Only rarely are calbindin-positive neurons found near the ventricles. Calbindin immunostaining of chimeric cerebellum (B) indicates that, although correctly located Purkinje cells are calbindin-positive, there are many ectopic Purkinje cells (ep) found within the parenchyma of the cerebellum near the regions of the deep cerebellar nuclei (dcn). No calbindin-positive neurons are apparent within the internal granule cell layer. In wild-type mice, there is complete congruence between calbindin-positive (C) and Cdk5-positive (D) neurons. Within the region of the Purkinje cell layer, there are some large neurons in both wild-type and chimeric mice that are Cdk5-positive but not calbindin-positive. These are probably other large neurons of the cerebellar cortex, such as basket, Lugaro, or Golgi cells. All calbindin-positive neurons (E) within the Purkinje cell layer of chimeras are wild type, i.e., Cdk5-positive (F). However, calbindin-positive Purkinje cells in the chimera (ectopically located near the ventricle in the top left corner ofG) are mutant, i.e., Cdk5-negative (H). Insets inG and H are higher magnifications of the areas indicated by the white boxes. Scale bars: (inA) A, B, 150 μm; (inC) C–H, 100 μm.

Fig. 4.

High magnification of deep cerebellar nuclei from wild-type (A, B) and chimeric (C, D) cerebella. Sections were double immunostained with calbindin (A, C) and Cdk5 (B, D). Wild-type deep cerebellar nuclei neurons can be identified by location and the presence of a calbindin-positive ring surrounding their cell body. The chimeric deep cerebellar nuclei contain both Cdk5-positive and Cdk5-deficient neurons. In the chimera, many deep cerebellar nuclei neurons contain Cdk5; however, some do not (arrowheads). Scale bar, 50 μm.

Fig. 5.

Cdk5 staining of granule cells in wild-type and chimeras visualized with confocal microscopy. Double immunostaining with antibodies β2/β3 GABA_A receptor (bd17) (Ewert et al., 1992) and Cdk5. The cell surface localization of bd17 (red) surrounds the Cdk5-positive (green) granule cell (Laurie et al., 1992;Fritschy and Mohler, 1995). Higher magnifications of boxed areas of A, C, andE are shown in B, D, andF, respectively. Granule cells are identified by membrane staining with bd17 and a diameter of 5–8 μm. The relative Cdk5 staining in granule cells is much lower than in Purkinjecells (A,top left corner, and C, bottom right corner). Granule cells of wild-type mice all contain Cdk5 (A, B). A few capillaries are stained by Cy3-conjugated anti-mouse IgG antibodies (arrowheads inA) and should not be confused with Cdk5-negative granule cells. The granule cell layer of the chimeric IGL contains bothcdk5^+/+ andcdk5^−/− neurons (C,D). These can be seen at high magnification inD, with cdk5^+/+granule cells indicated by the arrowheads andcdk5^−/− granule cells indicated byasterisks. The ectopic granule cell neurons of the chimeric molecular layer are Cdk5-deficient (E,F). Scale bar: A,C, E, 20 μm; B,D, F, 5 μm.

Fig. 6.

Northern blot analysis of mRNA expression forreelin, disabled-1(mdab-1), and GAPDH in E16.5 brains ofcdk5^+/+ andcdk5^−/− mice reveals no difference between wild-type and mutant mice.

Fig. 7.

Expression pattern of reelin mRNA in sagittal section of cerebrum (A, C) and cerebellum (B, D) by in situ hybridization. Antisense probes (synthesized from rl-1) hybridized to E16.5 Cdk5^+/+(A, B) andCdk5^−/− (C,D) show normal levels and distribution ofreelin mRNA in the olfactory bulb (left,facing arrowheads), along with the marginal zone of the cerebral cortex (arrowheads along top). Note the reelin expression within the external granule cell layer of the cerebellum (arrowheads) of wild-type andCdk5^−/− mice (B,D, respectively). No significant hybridization is observed when sense probe is used (data not shown). Two different cDNA fragments (rl-1 and rl-2) were used as probes, and the same hybridization patterns were obtained (rl-2, data not shown). Scale bars: A, C, 500 μm; B,D, 800 μm.