Malformations affecting the cerebellar vermis are only poorly understood at the genetic level. Dandy-Walker malformations (DWM) are characterized by dilation of the fourth ventricle, anterior rotation of the cerebellum, vermis hypoplasia or agenesis, and hydrocephalus, and they may occur together with other abnormalities (1). Recent work identified a subset of DWM patients carrying interstitial deletions on chromosome 3, most of which were hemizygous for both ZIC1 and ZIC4 (2), transcription factors that promote proliferation of cerebellar granule cell precursors. Mice heterozygous for both Zic1 and Zic4 reproduce several DWM phenotypes, but show more modest effects on other characteristic features (2). Identifying genes with related phenotypes should help to clarify the developmental mechanisms behind such malformations.The nur12 mouse was identified as having an ataxic gait and cerebellar hypoplasia, including agenesis of the vermis, in a large chemical mutagenesis screen (3). Here, we use positional cloning and noncomplementation with a gene trap allele to identify the Ebf-and SMAD-interacting 30 zinc-finger protein, Zfp423 (also known as ROAZ or OAZ) as the gene mutated in nur12 mice. Tsai and Reed (4, 5) first described ROAZ in rat olfactory tissue as an Ebf1 (previously OLF-1, or O/E-1)-interacting protein identified in a yeast two-hybrid assay. Based on the overlapping temporal sequence of Zfp423 and Ebf1 expression in olfactory precursors, they proposed that their interaction maintains olfactory precursors in an immature state, whereas release of Ebf to form homodimers promotes terminal differentiation. Subsequent genetic manipulations indicated that Ebf family members promote neuronal differentiation coupled to cell cycle exit and migration out of germinal zones (6-10). Our results show that Zfp423 is required for proliferation and differentiation by precursor cells in the developing brain. We propose that by forming mutually exclusive complexes with other transcription factors, Zfp423 sharpens the response of precursor cells to external differentiation signals. The phenotypic similarities between nur12 mutant cerebellum and DWM may shed further light on a group of human disorders for which few genetic clues have been found. Results Ataxia and Brain Malformations in nur12Mice. Homozygous nur12 mice have ataxia and tremor [see supporting information (SI) Movie 1] and have a malformation of the cerebellum that is more severe at the vermis than the hemispheres (Fig. 1A) and a pronounced anterior rotation of the cerebellum within the posterior fossa (Fig. 1B). These features are characteristic of certain human hindbrain malformations, including DWM and cerebellar vermis hypoplasia (CVH), but are not common among ataxic mouse mutations. Among intercross progeny, the nur12 cerebellum can range from complete loss of vermis with somewhat smaller hemispheres to almost complete loss of the cerebellum. Within

Chemical mutagenesis of the mouse is ongoing in several centers around the world, with varying estimates of mutation rate and number of sites mutable to phenotype. To address these questions, we sequenced ‫6.9ف‬ Mb of DNA from G 1 progeny of ethylnitrosourea-treated mice in a large, broad-spectrum screen. We identified 10 mutations at eight unique sites, including six nonsynonymous coding substitutions. This calibrates the nucleotide mutation rate for two mutagenesis centers, implies significance criteria for positional cloning efforts, and provides working estimates of effective genetic target sizes for selected phenotypes.M UTAGENESIS experiments provide both novel direct measurement of the nucleotide substitution rate (Beier 2000;Kile et al. 2003). In particular, comparison mutations for biological studies and the means to estimate genomic parameters of the traits under of the nucleotide change rate to the rate of recovered phenotypic deviants should allow calculation of the efstudy. For example, the effective number of genes or the genome-wide target size for a particular phenotypic fective genome target size mutable to a given phenotype, without making assumptions about the distribution of muclass are of interest both for understanding the genetic architecture of a trait and for assessing the saturation table sites among distinct genes. Direct ascertainment of induced nucleotide changes also provides immediate acof mutable sites within a given screen. Estimates of these parameters depend on knowing the mutation rate cess to mutations of potential biological interest. Accurate measurement of the substitution rate is additionally within the experiment. Mutation rates, first defined by Muller, classically have been estimated by specific locus useful in evaluating candidate polymorphisms in positional cloning efforts. Brown and colleagues have pretests for failure to complement phenotype of a known mutation (Muller 1928; Russell 1951; Russell and viously estimated the rate of nucleotide change in a large chemical mutagenesis using heteroduplex detection by Montgomery 1982). Such estimates can be biased by the nature of the chosen gene (or genes) and the generdenaturing HPLC as a surrogate for sequence (Coghill et al. 2002). These results may be difficult to apply more ality of phenotypes caused by its mutation. Inferring the nucleotide substitution rate from specific locus data generally because few mutations were observed (and the detection rate was not established), the potential therefore requires assumptions about the fraction of sites mutable to the tested phenotype and is open to subbias from sampling only three loci is unclear, and the mutagenesis protocol and strain choice are substantially stantial locus-specific bias. For many applications, the specific locus mutation rate is a sufficient term by itself, different from those used in other centers. Here we use DNA sequencing at 84 sentinel sites across 51 autosomal irrespective of the underlying nucleotide change rate. Indeed, classical esti...

Endogenous retroviruses have shaped the evolution of mammalian genomes. Host genes that control the effects of retrovirus insertions are therefore of great interest. The Modifier-of-vibrator-1 locus controls level of correctly processed mRNA from genes mutated by endogenous retrovirus insertions into introns, including the pitpn vb tremor mutation and the Eya1 BOR model of human branchiootorenal syndrome. Positional complementation cloning identifies Mvb1 as the nuclear export factor Nxf1, providing an unexpected link between mRNA export receptor and pre-mRNA processing. Population structure of the suppressing allele in wild M. m. castaneus suggests selective advantage. A congenic Mvb1 CAST allele is a useful tool for modifying gene expression from existing mutations and could be used to manipulate engineered mutations containing retroviral elements.Mus musculus is a complex species group with subpopulations that have diverged and hybridized since becoming commensal with humans some 10,000 years ago. Allopatric divergence and re-hybridization of mouse lineages are thought to contribute to the diversity and activity of retroviral elements in the current mouse genomes 1,2 . Host genes that can NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript influence the expression of newly introduced (or newly mobilized) viral elements might be selected for variants that blunt these effects. Our results suggest that Mvb1 is such a locus.Mvb1 was originally identified as a strain-derived locus that modifies the neurological mutant, vibrator 3. The vibrator (vb) mutation is a hypomorphic allele of the phosphatidylinositol transfer protein α (PITPα) gene (pitpn) caused by the insertion of an endogenous retrovirus (intracisternal A particle, or IAP) into the fourth intron of the gene, resulting in 5 to 10-fold loss of PITPα expression. Homozygous vibrator mice show severe action tremor, progressive degeneration of interneurons in the brain stem and spinal cord, and uniform juvenile lethality. However, vibrator mice carrying Mvb1 alleles from the wild-derived CAST/Ei inbred strain show reduced tremor severity and survive to adulthood. In principle, this could be due to a change in physiological requirement for PITPα function or to a change in the steady-state expression level of PITPα derived from the mutant allele. RESULTS Mvb1 is a dosage-sensitive modifier of vibrator RNA accumulationMvb1 modifies the severity of vibrator tremor and the associated juvenile lethality 3 . To examine this effect in more detail, we monitored the longevity of vb mutant mice congenic on a C57BL/6J (B6) strain background with zero, one or two CAST/Ei alleles of Mvb1 ( Figure 1a). Consistent with our behavioral observations, the longevity data indicate a semi-dominant mode of action and high penetrance.To extend these observations to the cellular level, we examined histology of vibrator animals homozygous for either the B6 or CAST allele of Mvb1 (Figure 1b To ask whether Mvb1 acts by altering RNA levels from the mutant...

Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1CAST suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including Ap3d1mh2J, a model for Hermansky-Pudlak syndrome, and Atcayhes, an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes ∼two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1CAST alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.

Interpreting rare variants remains a challenge in personal genomics, especially for disorders with several causal genes and for genes that cause multiple disorders. ZNF423 encodes a transcriptional regulatory protein that intersects several developmental pathways. ZNF423 has been implicated in rare neurodevelopmental disorders, consistent with midline brain defects in Zfp423-mutant mice, but pathogenic potential of most patient variants remains uncertain. We engineered~50 patient-derived and small deletion variants into the highlyconserved mouse ortholog and examined neuroanatomical measures for 791 littermate pairs. Three substitutions previously asserted pathogenic appeared benign, while a fourth was effectively null. Heterozygous premature termination codon (PTC) variants showed mild haploabnormality, consistent with loss-of-function intolerance inferred from human population data. In-frame deletions of specific zinc fingers showed mild to moderate abnormalities, as did low-expression variants. These results affirm the need for functional validation of rare variants in biological context and demonstrate cost-effective modeling of neuroanatomical abnormalities in mice.