Mouse Zic1 is involved in cerebellar development

Abstract

Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, we have generated mice deficient in Zic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.

Fig. 1.

Mouse Zic1 gene, targeting construct, mutated Zic1 gene, and verification of the targeted insertion. A, The wild-type mouseZic1 gene (middle) consists of three exons (Aruga et al., 1996a). The first contains the initiator methionine and three of five C2H2 type zinc finger motifs. The targeting vector (top) contains 8 and 2.4 kb regions homologous to the Zic1 gene and a neomycin resistance gene, respectively, driven by the PGK promoter (NEO). The diphtheria toxin A fragment gene driven by the MC1 promoter (DT) (Yagi et al., 1993) was inserted in the 3′ end of the Zic1 gene to eliminate nonspecific integration. In a properly mutated allele (bottom), the protein coding region and splicing donor site of the mouseZic1 gene exon 1 has been replaced by the neomycin resistance gene expression unit. B,EcoRI-digested genomic DNA samples extracted from a litter were analyzed by Southern blotting using three probes (5′, 3′, NEO). The band size and the corresponding fragments are indicated A and B, respectively. B, BglII; R, EcoRI; S,SalI; X, XhoI. C, Viability of Zic1−/− mice. Survival rates of 13Zic1−/− mice with the C57BL N2 background and 13 with the C3H N2 background were determined up to postnatal day (PND) 21. Pups in each group were derived from at least five litters. There were no deaths among Zic1+/+ andZic1+/− littermates during the experimental period.

Fig. 2.

Abnormal behavior was observed in theZic1−/− mice. A,Zic1−/− mice (P10) (right) often lay on their backs in infancy, whereas littermates (left) did not. The mouse distorted its body in an attempt to turn over but was unable to do so. When the mice were suspended by their tails, theZic1−/− mice (P14) often crossed or clasped their hindlimbs (C), whereas the normal mice usually extended and shook their hindlimbs (B).

Fig. 4.

Morphometric analyses of midsagittal sections of mature cerebella. A, Areas of the granular cell layer (GCL) and molecular layer (ML).B, Perimeter of the Purkinje cell layer in both midsagittal (mid) and cerebellar hemisphere (lat) regions. C, Ratios of anterior GCL area (rostral to primary fissure) to posterior GCL area (caudal to primary fissure). Granule cell density (D) and Purkinje cell number per millimeter (E) in midsagittal regions were measured in Zic1+/+ (open bar), Zic1+/− (hatched bar), and Zic1−/− (shaded bar) cerebella. Error bars indicate SEM. *p < 0.01; **p < 0.001.

Fig. 5.

In situ hybridization of tissues from Zic1+/+ (A, C) orZic1−/− (B, D) cerebella. In situ hybridization using En2 (A, B) and Zic2 (C, D) probes showed the expression of these genes to be essentially unchanged in theZic1−/− cerebella. The cerebella were taken from C3H N1 mice. Scale bar, 1 mm.

Fig. 6.

Phenotypes of the developing cerebella. A series of sagittal sections through the midsagittal planes of theZic1+/+ (A–C, G) orZic1−/− (D–F, H) cerebella were stained with hematoxylin–eosin. E17.5 (A, D), E18.5 (B, E), and P5 (C, F) cerebella were examined. CP, Choroid plexus; T, tectum; IV, fourth ventricle; e, external germinal layer; closed arrowheads, lucent portions indicating the cessation of the immature Purkinje cell layer (asterisk); open arrowheads, primary fissures. G, H, Higher magnifications ofC and F, respectively. e, External germinal layer; g, granule cell layer;m, molecular layer; p, Purkinje cell layer. Note that the Purkinje cells of Zic1−/− cerebella are morphologically immature at this time point. All panels indicate the C3H N1 cerebella. Scale bars: A–C, 200 μm; G, 100 μm.

Fig. 7.

BrdU labeling study of developing cerebella.A, BrdU-labeled cells in the EGL, on sections of the cerebellar sections anlage, were counted. B, Change in EGL length in the developing cerebella. Rostral to caudal lengths of the EGL were measured at each developmental stage. In Aand B, all data for Zic1−/− are significantly different from those for Zic1+/+ cerebella (p < 0.001, t test).C, BrdU-labeled cells are compared in a lengthwise portion. Open and closed bars(circles) indicate the Zic1+/+ andZic1−/− cerebella, respectively. From E14 through E18, the data for Zic1−/− cerebella are significantly different from those for Zic1+/+ cerebella (p < 0.001, paired t test). The data are presented as means. Error bars indicate SEM. *p < 0.01; **p < 0.001.