Zfp423 is required for normal cerebellar development

Abstract

Zinc finger protein 423 (also known as Ebf-associated zinc finger protein, Ebfaz) binds to and negatively regulates Ebf1, a basic helix-loop-helix transcription factor required for B-cell lineage commitment and olfactory epithelium development. Zfp423 also binds to Smad1/Smad4 in response to Bmp2 signaling. Zfp423 contains 30 Krüppel-like zinc fingers that are organized into discrete clusters; some zinc fingers are used to bind DNA, while others mediate Zfp423's interaction with other signaling proteins such as Ebf1 and Smad1/Smad4. Previously, we showed that Zfp423 is an oncogene whose upregulation following retroviral integration in murine B cells leads to an arrest in B-cell differentiation and the subsequent development of B-cell lymphomas. To study the biological functions of Zfp423 in vivo, we used recombineering and gene targeting to generate mice that carry conditional as well as null alleles of Zfp423. Homozygous Zfp423 null mice are runted and ataxic, the cerebellum is underdeveloped, and the vermis is severely reduced. In the remaining cerebellar structures, the Purkinje cells are poorly developed and mislocalized. In mice carrying a hypomorphic Zfp423 gene trap allele, lacZ expression in the cerebellum correlates with the Purkinje cell layer, suggesting that these phenotypes are a result of a Purkinje cell-intrinsic defect.

FIG. 1.

Generation of null and conditional Zfp423 alleles. (A) Zfp423 genomic region encoding exon 4. Restriction fragment sizes are indicated as well as the positions of the internal and the two flanking probes used for genotyping analysis (roman numerals). The shaded area indicates the part of the genomic region included in the targeting vector, and the three different alleles described in this study are shown. neo is the result of the gene-targeting event. cko is the conditional knockout allele derived from the neo allele after Flpe-mediated excision of the PGK-neo cassette. One Frt site and two loxP sites remain in the locus. ko is the null allele derived from the neo or from the cko allele by Cre-mediated recombination between the two loxP sites. Only a single loxP site remains in the modified locus. loxP and Frt sites are indicated as black and red triangles, respectively. Neo, PGK-em7-neomycin dual selection cassette for bacteria and ES cells; TK, thymidine kinase cassette for counterselection in ES cells; H, HindIII; B, BglII; N, NotI. The genomic region is not drawn to scale. (B) Results of a Southern blot analysis of BglII-digested tail DNA, probed with the internal probe (II). The genotypes are indicated above each lane, and the sizes of the bands are indicated to the left. (C) Results of a poly(A) Northern blot analysis of whole-brain RNA from 3-week-old mice, using a full-length Zfp423 cDNA probe. After decaying, the blot was rehybridized with a Gapdh probe as a control for RNA quality. Genotypes are indicated as well as the positions of the different bands. wt, wild type.

FIG. 2.

Runted size and gross cerebellar defects in Zfp423 null mice. (A) Photograph showing the size difference between two 5-week-old male littermates. The genotypes are indicated. (B) Photographs of paraformaldehyde-perfused and fixed brains clearly showing the severely reduced cerebellar vermis in two 13-day-old Zfp423 mutants. All four pictures are shown to the same scale. Ve: vermis; He: hemisphere.

FIG. 3.

Cerebellar defects in postnatal development in Zfp423 mutant and control mice. Mid-sagittal H&E-stained cerebellar sections from two littermates from four different stages of postnatal development (P4, P7, P10, and P13), clearly showing the decreased cerebellar size and reduced foliation. All pictures are shown to the same scale.

FIG. 4.

Cerebellar defects in 1-year-old Zfp423 mutant mice. H&E-stained sections from three female (A) or three male (B and C) littermates of 1 year of age. (A) Mid-lateral sagittal sections of the cerebellum showing that the reduced cerebellar size is still evident at 1 year of age. All sections were cut at the same distance from midline and shown at the same magnification. (B) Frontal half-cerebellar sections from three 1-year-old male littermates. Note the severe reduction in size of the cerebellar vermis and clearly reduced cerebellar size. All sections were cut at the same distance from the cerebellar/midbrain border and shown at the same magnification. He, hemisphere; Ve, vermis. (C) Higher-power magnification of the cerebellar cortex; all sections are shown at the same magnification. ML, molecular layer. White arrows point to the presence of some of the ectopic Purkinje cells in the molecular layer in the ko/ko mutant.

FIG. 5.

Subtelecephalic structures of the Zfp423 null brain are smaller than those in normal littermates. H&E-stained sagittal sections of brains from ko/ko and normal (ko/+ or +/+) littermates were collected from E15 to 1 year of age. Note the normal size of the olfactory bulb and cerebral cortex at all stages of development. Met/myelencephalic structures are hypomorphic, however, with the cerebellar dysgenesis most prominent. One-millimeter scale bars are indicated. Asterisks indicate enlarged ventricles in the mutant brains. hb, hindbrain.

FIG. 6.

Misorganization of Purkinje cells in Zfp423 ko/ko mice. Calbindin (red)- and DAPI (blue)-stained sagittal cerebellar sections from four different stages of postnatal development, as indicated, analyzed by confocal microscopy. Note the developmental delay of Purkinje cells as well as the lesser degree of dendritic branching and general Purkinje cell body misorganization. EGL, external granule cell layer. The genotypes are indicated.

FIG. 7.

XH542 is a hypomorphic allele of Zfp423. (A) Intron 3, where the βgeo reporter (blue box) is integrated. Both the wild-type (WT) and gene trap alleles are shown. Exons 3 and 4, the BglII sites (B), the genomic probe used for genotyping by Southern blot analysis, and the sizes of the restriction fragments are indicated. The sources of the three detectable splice products are shown: exon 3-exon 4 splicing (wild type; green); exon 3-βgeo splicing (red), and the composite aberrant splicing event (exon 3-plasmid-exon 4; blue). The positions of the three primers used for RT-PCR are also indicated. SA, engrailed splice acceptor. The genomic map is not drawn to scale. (B) Genotyping by Southern blot analysis. gt, gene trap allele. (C) Results of RT-PCR analysis using the forward primer located in exon 3 together with a reverse primer in exon 4 (upper panel) or in lacZ (lower panel). The presence of a wild-type product in the absence of a wild-type allele clearly shows that the wild-type messenger is being made from this allele. (D) Mid-sagittal sections (heterozygous gt/+ mice) from embryonic day 15.5 and postnatal day 4, stained for lacZ expression and counterstained with neutral red. The position of the external granule layer (EGL) is indicated. lacZ staining correlates with the expected position of Purkinje cells. (E) H&E-stained mid-lateral sagittal sections from adult mice of the indicated genotypes. Note the hypomorphic phenotype of the gt/ko brain. All sections were cut at the same distance from the midline.