Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions

Abstract

Members of the En and Wnt gene families seem to play a key role in the early specification of the brain territory that gives rise to the cerebellum, the midhindbrain junction. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, we expressed En-2 ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity to the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures that is highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions.

Fig. 1.

Construction and characterization of L7En-2 transgenic mice. A, Gene structure of the endogenous L7 gene (top), the L7En-2 transgene (middle), and the L7β-Gal construct (bottom). The promoter of the L7En-2 transgene is ∼1 kb; that of L7β-Gal (corresponding to L7βG3 that expresses bands in both a precocious and prolonged manner; Oberdick et al., 1993) is ∼0.5 kb. Translation of the L7En-2 transgene construct can only initiate within the En-2 coding region because all possible start ATGs were removed from the L7 exons to form the L7ΔAUG base vector (Smeyne et al., 1995). Animals carrying the L7β-Gal construct were crossed into L7En-2 mice to test the effect of En-2 on sagittal banding (see Fig. 4). B, Northern blot analysis to detect L7En-2 transgene expression. The panel on theleft was probed with L7 cDNA and that on theright with En-2 (using the same fragment used forin situ hybridization; see Materials and Methods). Although from the same gel, the two panels represent different size ranges (note arrows denoting positions of 18S RNA). In both panels, L7En-2 refers to the mRNA encoded by the transgene. Note that the level of transgene mRNA is significantly lower than is that of endogenous L7 (left) but is equal to the level of endogenous En-2 (right).wt, Ten micrograms of wild-type cerebellar RNA;tg, 10 μg of transgenic cerebellar RNA.C, Detection of correct L7 and En-2 protein species in L7En-2 transgenic cerebellum by Western blot. No aberrant-sized proteins were detected with either L7 or En-2 antibodies as might result from unexpected frameshifts or L7-En-2 protein fusions.wt, Twenty micrograms of wild-type cerebellar protein;tg, 20 μg of L7En-2 cerebellar protein.

Fig. 2.

Detection of transgene expression in Purkinje cells by in situ hybridization and immunocytochemistry.A–D, In situ hybridization with a 218 bp³⁵S-labeled antisense riboprobe corresponding to the N-terminal portion of the En-2 coding region is shown. En-2 expression in the wild type is restricted to the granule cell layer (A, C). In L7En-2 transgenics, En-2 is detected in granule cells and Purkinje cells (arrowheadsin B, D). E,F, En-2 protein is restricted to granule cells in P3–P4 wild-type cerebellum (E) but is additionally expressed in Purkinje cell nuclei (arrowhead) in L7En-2 transgenics (F). pcl, Purkinje cell layer; gcl, granule cell layer. Scale bars:A, B, 500 μm; C,D, 100 μm; E, F, 50 μm.

Fig. 3.

Embryonic and neonatal expression of L7En-2 transgene. A, RT–PCR was performed using RNA prepared from E13, E14.5, P0, and adult cerebellum. Primers were designed that could distinguish the L7En-2 transgene mRNA from endogenous L7 and En-2 mRNAs (see Materials and Methods). Whereas En-2 mRNA is detectable at all time points and in both transgenics (tg) and wild types (wt), L7 is only very weakly detectable in both genotypes at E14.5 but is strongly detectable at P0 and in adults. The transgene mRNA can only be detected in P0 and adult transgenics, never in wild types. B–D, In situ hybridization to detect L7En-2 transgene expression is shown. Horizontal sections of E17.5 wild-type (B, C,left) and transgenic (right) cerebellum were hybridized with En-2 probe (B) or L7 probe (C). The En-2 probe reveals a pattern of expression in the transgenic that is a superimposition of the wild-type L7 and En-2 expression patterns. Arrowheads indicate positions of endogenous En-2 clusters and L7-negative zones. An oligonucleotide probe (D) sequences (see Materials and Methods) was used to compare transgene expression with endogenous L7 expression. Transgene is undetectable in wild types (D, left) but is detectable in transgenics (middle) in a pattern identical to that of L7 in adjacent sections (right).

Fig. 4.

Analysis of L7 sagittal bands in L7En-2 transgenic mice. Animals expressing the L7βG3 transgene were crossed with L7En-2 mice, and the β-gal banding pattern was compared with that in a wild-type background. Images are whole-mount views of rostral (A) and caudal (B) cerebellar aspects at P11. In both A andB, the wild type is at the top, and the mutant is on the bottom. The arrowhead inA indicates the fissure dividing lobuleVI into a and bsublobules; this fissure is missing in the transgenics. Similarly, the fissure separating IXa and IXb is deleted in the transgenic (B). Roman numerals identify selected lobules.

Fig. 5.

Change in lobulation pattern in L7En-2 cerebellum.A, C, Midline sagittal sections of wild-type (A) and L7En-2 mutant (C) cerebella. B,D, Lateral sections from the same brains shown inA and C. All sections were stained with methyl green. Note the loss of the fissure dividing sublobulesVIa and VIb in the mutant, shown in whole mount in Figure 4. This loss persists into the hemispheres as a loss of the fissure subdividing the lobule simplex. Arrowsindicate the sublobule fissures other than that in VIthat are deleted in the mutant. Scale bar, 500 μm. Roman numerals identify vermal lobules. Sim, Simplex lobule; C1, crus I of ansiform lobule;C2, crus II of ansiform lobule; PM, paramedian lobule.

Fig. 6.

Gaps in the Purkinje cell layer within central lobe fissures. A–E, Sagittal sections near the midline were prepared for immunocytochemistry with antibodies against the L7 protein. Stunted dendritic morphologies (B) characterize the Purkinje cells in fissures at P7, as compared with their normal appearance at this age in wild types (A). These stunted morphologies give way in the central lobe fissures to cell-sparse patches of Purkinje cells that adopt tangential dendritic orientations (C). Gaps within the central lobe fissures are also evident in the transgenic (E, arrowheads) but not in the wild type (D), especially in the fissure-dividing lobulesVII and VIII. F, By P5, when the stunted-Purkinje cell dendritic morphology is first seen, transgene expression is uniform (as revealed with antibodies to En-2). Thus the fissure-restricted phenotypes described here are attributable to a selective sensitivity of these cells to transgene expression. Scale bars: A–C, 50 μm;D, E, 100 μm; F, 300 μm.

Fig. 7.

Retarded EGL formation within cerebellar deep fissures. A, B, Sagittal sections of P7 cerebellum from wild type (A) and L7En-2 mutant (B) showing a thinned EGL in the vermal fissures of the mutant. Sections were stained with methyl green. Note the lack of fusion of the two sides of the EGL at the base of the fissures (arrows in B). Scale bar, 200 μm.C, D, Comparison of the EGL thickness in wild-type versus L7En-2 transgenic cerebellum in the vermis (C) and hemispheres (D). The EGL within wild-type and mutant cerebellar sections was divided into an equal number of segments, and its thickness along the entire cerebellar surface was determined. Note that at the base of every fissure, the thickness of the mutant EGL was thinner than in the wild type, and this effect is maximized in the fissures of the central lobe [V–VI (primary fissure), VI–VII, and VII–VIII]. The twocurves of each plot tend to converge at the summit of the lobules and to diverge within fissures. The values are averages of three near-midline sections and three lateral sections calculated for one cerebellum of each genotype, and these same observations were repeated in two additional P7 cerebella. Abbreviations along thex-axis in D are defined in Table 2.