ENU mutagenesis in the mouse COMMENT Outlook Finding the genes that direct mammalian development ENU mutagenesis in the mouse The genetic control of mammalian embryogenesis is not well understood. N-ethyl-N-nitrosourea (ENU) mutagenesis screens in the mouse provide a route to identify more of the genes that are required for mammalian development. The characterization of ENU-induced mutations can build on the resources provided by the mouse and human genome projects to help define the tissue interactions and signaling pathways that direct early mammalian development. he mouse has a rich tradition in developmental genetics. implantation, survival of the mammalian embryo depends TMany of the genes that have provided the special insights on the development of a series of physical connections into mammalian development were discovered on the basis between the developing embryo and maternal circulation, of the phenotypes of mutant mice1 (Table 1). For example, including the placenta, that have no counterpart in Brachyury, the founding member of the Tbox gene family, invertebrate or fish embryos. In addition, analysis of the was identified in the 1920s because Brachyury (T)/1 mice Zebrafish genome has led to the surprising finding that have short tails; shortly thereafter it was found that T/T there are seven Hox clusters in Zebrafish in contrast to the homozygotes die at midgestation and the posterior part of four clusters in mammals3, suggesting that there might the body axis is truncated2. have been an extra genome duplication in the teleost In the recent past, the strengths of classical mouse lineage4. Thus, there will be no simple relationship genetic studies have been overshadowed, both by targeted between the phenotypes of mouse and fish mutants in mutagenesis in the mouse and by model organism genetics. homologous genes. Targeted mutagenesis currently dominates mouse genetics Despite the major contributions of model organisms because of its extraordinary ability to reveal the function and gene targeting, mutations have been made in <5% of of cloned genes. At the same time, the ease of large-scale the genes in the mouse genome. Recent work suggests that genetic screens in Drosophila, Caenorhabditis elegans and ENU mutagenesis screens have the potential to return Zebrafish has made these organisms popular for phenotype- classical mouse genetics to the forefront of mammalian gene based gene discovery. However, mammalian development discovery. An arsenal of reagents to characterize mouse differs in fundamental respects from the development of genes identified by mutant phenotypes is now available, all these model organisms. For example, transcription including bacterial artificial chromosome (BAC) transgenics in mammalian embryonic nuclei begins days before for mutant complementation and integrated genetic and gastrulation, so the critical events of germ-layer determi- physical maps for positional cloning. This article focuses nation and early morphogenesis depend on zygotically on ENU-mutagenesis approaches to identify genes that transcribed genes rather than on maternal stores. After control mouse development. TABLE 1. Examples of mouse developmental regulatory genes identified on the basis of mutant phenotypes Gene Origin Phenotype Protein encoded Refs (cloning approach) brachypodism Spontaneous recessive Skeletal defects in limbs Gdf5 (candidate gene) 20 Brachyury (T ) Spontaneous dominant visible: Truncated axis T (positional cloning) 2, 21 short tail eed ENU-induced lethal over albino Lack of anterior development (null); esc homolog; Polycomb-group gene 10 deletion homeotic transformation (hypomorph) (positional cloning in region defined by deletion breakpoints) Extra-toes Spontaneous dominant visible: Neural tube and limb defects Gli3 (candidate gene) 22 polydactyly Fused/Axin Spontaneous dominant visible: Axis duplications Axin (transgene insertion allele) 23 kinked tail kreisler Spontaneous recessive Circling behavior; hindbrain abnormal Transcription factor (positional cloning) 24 Kathryn V. Anderson nodal Retroviral insertion-induced: No mesoderm produced Nodal (BMP family member) (retroviral 25 k-anderson@ recessive lethal insertion allele) ski.mskcc.org short ear Spontaneous recessive Skeletal defects Bmp5 (positional cloning within deletion region) 26 situs inversus viscerum (iv) Spontaneous recessive Randomization of left–right asymmetry Left–right dynein (transgene insertion allele) 27 Splotch Spontaneous dominant visible: Neural tube and neural crest defects Pax3 (candidate gene) 28 Molecular Biology white belly spot Program, Sloan-Kettering Institute, 1275 York Abbreviations: Bmp5, bone morphogenic protein 5; eed, embryonic ectoderm development; esc, extra sexcombs; Gdf5, growth differentiation factor 5; Gli3, Gli-Kruppel family member Gli3; Avenue, New York, Pax3, paired box gene 3 product. NY 10021, USA. 0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(99)01921-6 TIG March 2000, volume 16, No. 3 99 Outlook COMMENT ENU mutagenesis in the mouse TABLE 2. Examples of mouse developmental mutants identified the most efficient ENU dosage regime gave an average of one new mutation per gene in only 700 first-generation a in ENU mutagenesis screens (F1) progeny in seven loci that produce visible phenotypes (the specific locus test)5. Assuming that there are ~70 000 Screen Examples of mutations identified Refs genes in the mouse genome, each F1 animal is heterozygous Lethals in trans to albino deletions eed (3 alleles); five additional prenatal lethal loci 7 for ~100 new inactivating mutations. This high efficiency of Lethals in T/t region quaking (4 lethal alleles); new alleles of T; 29– 31 mutagenesis makes it possible to identify the small fraction nine additional prenatal lethal loci of genes that affects any particular biological process. For Failure to complement the behavioral kreisler allele 24 defect of a spontaneous example, in a screen of only 100 ENU-treated lines, kreisler allele ~10 000 different new mutations can be tested. Dominant behavioral mutants Wheels (inner ear defects) 14 Morphological abnormalities at 5 embryonic lethal mutants 11 Region-based screens midgestation (9.5 dpc) Forebrain defects at midgestation flat-top; 4 other mutants 12 The potency of ENU as a mutagen opens up a variety of (10.5 dpcb) approaches to identify genes that play important roles in mammalian development (Table 2). One strategy is to aN-ethyl-N-nitrosourea. look for mutations in a defined region of the genome. The b dpc: days post-coitum. simplest version of this type of screen is to identify lethal mutations that fail to complement a particular chromosomal ENU: an extraordinarily efficient mutagen in the deletion (Fig. 1a). For example, 24 ENU-induced mouse mutations that are lethal when in trans to particular albino Efficient chemical mutagenesis in the mouse using the point deletions have been identified in large-scale experiments6,7. mutagen ENU was developed more than 20 years ago. The It is interesting to note that only one of the genes defined unique strength of ENU mutagenesis is its great efficiency: in these screens (eed) is known to be homologous to a gene FIGURE 1. Two kinds of screens to identify recessive mutations that affect embryonic development of the mouse (a)ENU (b) ENU female male female male (wild-type coat) +/+ c /c (albino coat) +/+ +/+ ch F1 c */+ c / Deletion (c ) F1 */+ +/+ F1 female male (chinchilla coat) F1 male females G2 G2 */+ ch 50% c / Deletion (c) ch * c /c +/c and +/Deletion (c) * female (albino) (chinchilla) (wild-type coat) progeny 25% 25% 50% Viable? (carriers) G3 Embryos 25% */* Phenotype? trends in Genetics An asterisk represents a newly induced mutation. (a) A region-based screen6. The males that are treated with ENU are homozygous for a recessive point mutant allele of albino and hence have albino-colored coats. Single F1 animals, which are each heterozygous for a c chromosome that might carry new mutations, are mated with animals that are heterozygous for a weak allele of c (cch ) and an albino (c) deletion, which removes the albino locus and a set of flanking genes. Newly induced lethal mutations in genes near albino are identified in the G2 generation because there are no viable albino (pure white) mice, which would have been heterozygous for the c deletion and the mutagenized c chromosome. The surviving mice with chinchilla colored coats carry the mutagenized c chromosome in trans to the cch chromosome; these animals are carriers that can be bred to perpetuate the new recessive lethal mutation. Once a line of animals that are heterozygous for the lethal mutation is established, whether the lethal mutation produces an interesting developmental phenotype can be tested. (b) A genome-wide screen for recessive embryonic lethal mutations that produce a clear morphological phenotype9. Single F1 males, which are each heterozygous for many different newly induced mutations distributed throughout the genome, are mated to produce a number of G2 females. Each G2 female has a 50% chance of inheriting any single mutation carried by the F1 male. If she does carry a particular mutation, then 25% of the embryos from the cross between that G2 female with her father will be homozygous for the mutation. In this scheme, mutations are identified on the basis of abnormal morphology at a particular developmental stage, rather than being first identified on the basis of homozygous lethality. Mapping of mutations using polymorphic simple sequence