Focusing Forward Genetics: A Tripartite ENU Screen for Neurodevelopmental Mutations in the Mouse

Abstract

The control of growth, patterning, and differentiation of the mammalian forebrain has a large genetic component, and many human disease loci associated with cortical malformations have been identified. To further understand the genes involved in controlling neural development, we have performed a forward genetic screen in the mouse (Mus musculus) using ENU mutagenesis. We report the results from our ENU screen in which we biased our ascertainment toward mutations affecting neurodevelopment. Our screen had three components: a careful morphological and histological examination of forebrain structure, the inclusion of a retinoic acid response element-lacZ reporter transgene to highlight patterning of the brain, and the use of a genetically sensitizing locus, Lis1/ Pafah1b1, to predispose animals to neurodevelopmental defects. We recovered and mapped eight monogenic mutations, seven of which affect neurodevelopment. We have evidence for a causal gene in four of the eight mutations. We describe in detail two of these: a mutation in the planar cell polarity gene scribbled homolog (Drosophila) (Scrib) and a mutation in caspase-3 (Casp3). We find that refining ENU mutagenesis in these ways is an efficient experimental approach and that investigation of the developing mammalian nervous system using forward genetic experiments is highly productive.

The Scribble–Dlg–Lgl Module in Cell Polarity Regulation

“…Mice homozygous for Scribble mutant alleles, including circletail (Crc), and the ENU mutants rumpelstiltzchen (rumz), crn2, and 5120-6B, or an engineered null mouse, all display multiple failures of developmental migration processes resulting in severe neural tube closure defects (craniorachischisis), abdominal wall defects, and an "eyes-open-at-birth" phenotype, which are all phenotypes characteristic of defects in PCP mutants (Murdoch et al 2003;Zarbalis et al 2004;Wansleeben et al 2011;Stottmann et al 2011;Pearson et al 2011). Interestingly, mutations in Scribble are associated in humans with the most severe type of neural tube closure defects, craniorachischisis, as well as spina bifida (Robinson et al 2012;Lei et al 2013).…”

“…4.2b). Mice mutant for Scribble display the characteristic PCP mutant phenotype, namely, neural tube closure defects, cochlear hair cell orientation defects, and "eyes-open-at-birth" phenotypes (Montcouquiol et al 2003;Murdoch et al 2003;Zarbalis et al 2004;Pearson et al 2011;Wansleeben et al 2011;Stottmann et al 2011). In addition, Scribble interacts both physically and genetically with the core PCP protein Vangl2 during neural tube closure and in the development of branched structures such as the lung (Yates et al 2013;Montcouquiol et al 2003).…”

ENU Mutagenesis in the Mouse

“…The utility of this reagent for generating mutations in the mouse was clearly shown by Russell et al (1979); however, because it was known to result in single nucleotide changes that were perceived as difficult to identify, it was originally employed by only a small cohort of intrepid investigators (Bode, 1984; Shedlovsky et al, 1986). As the tools for genomic analysis and positional cloning have been refined, the potential of ENU mutagenesis has become increasingly appreciated, and it has been employed both in wide-ranging screens involving large numbers of mice (Hrabe de Angelis, 2000; Nolan, 2000) and in smaller efforts focused on specific biological questions (Herron et al, 2002; Zarbalis et al, 2004; Huangfu and Anderson, 2005; Stottmann et al, 2011; Ha et al, 2013). Results of these efforts confirm the efficiency of the approach for producing novel mutations with interesting phenotypes.…”

SeqAnt 2012: Recent Developments in Next-Generation Sequencing Annotation

“…Forward genetic screens in Mus musculus have been very informative, revealing unsuspected mechanisms governing basic biological processes [27][28][29][30][31][32]. In this approach, a potent chemical mutagens, such as N-ethyl-N-nitrosourea (ENU), is used to randomly induce mutations in mice.…”