Pattern deformities and cell loss in Engrailed-2 mutant mice suggest two separate patterning events during cerebellar development

Abstract

Null alleles of the mouse Engrailed-2 gene, a molecular homolog of the fly gene engrailed, have demonstrable effects on the anteroposterior (A/P) patterning of cerebellum as reflected in the disruption of the normal process of foliation of the cerebellar cortex and the alteration of transgene expression boundaries in the adult. Engrailed-2 also affects the transient mediolateral (M/L) pattern of En-1 and Wnt-7b expression seen in late embryogenesis. We have examined three markers of cerebellar compartmentation in En-2 mutant mice: the Zebrin II and Ppath monoclonal antibodies and the transgene L7lacZ. In En-2 mutants, the normal temporal pattern of expression is preserved for all three markers, although the size and spatial location of various bands differ from those of the wild type. Unlike the foliation abnormalities, the M/L pattern disturbances we have found occur in nearly all cerebellar regions. Cell counts reveal that all major cell types of the olivocerebellar circuit are reduced by 30-40%. We propose that these results are best explained by a model in which the Engrailed-2 gene is involved in the early specification of the cerebellar field including the number of progenitors. Because each of these progenitors gives rise to a clone of defined size, Engrailed-2 helps specify adult cell number. We further postulate that the configuration of the seven Zebrin bands as well as the shapes and locations of the cerebellar lobules are set up by a second patterning event that occurs after neurogenesis is complete.

Fig. 1.

Sagittal view of wild-type andEn-2 mutant cerebella cresyl violet-stained midline (A, B) and lateral (C, D) sections from wild-type (A, C) and En-2^hd/hd(B, D) animals. Note the posterior folial abnormalities in lobules 8 and 9 at the midline (compare B with A) and the fusion of theCrus II and pml lobules in the hemisphere (compare D with C). Scale bar, 500 μm. pml, Paramedian lobule; simp, simplex.

Fig. 2.

Lack of En-2 function results in the fragmentation or loss of Zebrin II bands in the adult. Zebrin II bands in wild-type (A, C, E) andEn-2^hd/hd (B, D, F) cerebellar cortex are identified according to the nomenclature ofHawkes and Gravel, 1991. Sections are from posterior lobules VIII and IX (A, B), dorsal lobule V (C, D), and anterior lobule III (E, F). Note the shortening of the mutant P2 and P3 bands in the posterior lobules on theleft (up-facing brackets in A, B) and the fragmentation of the same bands on the right (down-facing bracket). In lobule V (C, D) there is a similar shortening of the P2 bands bilaterally, whereas in lobule III (E, F) the P2 band is missing in the mutant. Scale bar, 200 μm.

Fig. 3.

Reconstruction of coronal sections of Zebrin II- and Ppath-stained cerebellar cortex. The relative locations of the Zebrin II bands (filled lines) and Ppath interbands (stippled lines), as well as the folial differences between wild type (A) and theEn-2^hd/hd mutant (B) are illustrated in a representative coronal section. The hatched region in the mutant Crus I (C I) folium represents Purkinje cells reactive to both Zebrin II and Ppath. The Ppath-positive interband between Zebrin II bands P5 and P6 is either missing or the cells present in this region have switched from Ppath to Zebrin II expression. CII, Crus II.

Fig. 4.

Developmental compartments are disrupted early in the En-2^hd/hd mutant. Whole-mount cerebella from En-2^hd/hd, L7lacZ(B, D, F, H) stained with β-galactosidase are compared with wild-type mice (A, C, E, G) at different perinatal ages: E18 (A, B), P0 (C, D), and P2 (E, F). At E18, two medial bands are present in both control (A) and mutant (B) animals. By P0, the first disruptions are apparent. In wild-type (C), four bands are located in the region of the eighth and ninth folia, compared with the highly diffuse staining (asterisk) found inEngrailed-2 mutants (D). At P2 (E–H) many differences are noticeable. There is a loss of a midline interband (F, asterisk), the precocious appearance of a stained band in Crus I (F, located under I in the mutant), and the addition of two medial bands in lobule IX (G, compare bandsa, b in wild type with the pattern in theEn-2^hd/hd lobules 8, 9,H). S, Simplex.

Fig. 5.

L7lacZ band loss persists into adulthood. Whole-mount cerebella from En-2^hd/hd,L7lacZ (B, D, F) stained with β-galactosidase are compared with wild-type mice (A, C, E) at different postnatal ages: P4 (A, B), P7 (C, D), and P30 (E, F). P4 mutants lack lateral bands of transgene activity in vermal lobules III and IV, and staining is generally diffuse (A, B, asterisks). By P7 staining in the Crus II- and pml-fused folia reveals that band width is significantly reduced in the mutant (compare the region identified by the asterisk in C, lobule8, with that in D). Severe band loss is recognizable in the adult mutant (F), most noticeably in the vermis compared with wild type (E). In general, there appears to be less overall reaction product in En-2 mutants at this time.S, Simplex; I, Crus I; II, Crus II.

Fig. 6.

A, Effect ofEngrailed-2 on neuronal cell number. The percentage of each cell type in wild-type (shaded bar, 100%) and affected (solid bar) animals is shown. All of the principal neurons of the olivocerebellar circuit are affected to about the same extent. Counts of the motor neurons of the facial nucleus serve as a control, indicating that the deficit is specific for the cell types shown. B, Uniform reduction of Purkinje cell number. The raw counts from hemicerebella of mutant and control animals are shown graphically (□, wild-type; •, En-2^hd/hd). That the overall shapes of the curves are nearly identical indicates the lack of regional variation in the Purkinje cell loss observed in the mutant. C, Purkinje cell loss in theEn-2 mutant cerebellum. In this diagram, Purkinje cell counts are displayed folium by folium. Each box is proportional in area to the number of Purkinje cells in the corresponding folium. The shaded region represents the the number of Purkinje cells found in each folium in theEn-2 mutant. In each case, about two-thirds of the box is shaded, indicating an approximate one-third loss of cells. This loss appears uniform, because it is not greater or lesser in any given folium.