Analysis of mouse embryonic patterning and INAUGURAL ARTICLE morphogenesis by forward genetics Marı´a J. Garcı´a-Garcı´a*,Jonathan T. Eggenschwiler*†, Tamara Caspary*‡, Heather L. Alcorn*, Michael R. Wyler*, Danwei Huangfu*§, Andrew S. Rakeman*¶, Jeffrey D. Lee*, Evan H. Feinberg*ʈ, John R. Timmer*,**, and Kathryn V. Anderson*†† *Developmental Biology Program, Sloan–Kettering Institute, 1275 York Avenue, New York, NY 10021; §Neuroscience Program and ¶Molecular and Cell Biology Program, Weill Graduate School of Medical Sciences, Cornell University, 445 East 69th Street, New York, NY 10021; and ʈWeill Graduate School of Medical Sciences at Cornell University͞The Rockefeller University͞Sloan–Kettering Institute Tri-Institutional M.D.-Ph.D. Program, 1300 York Avenue, New York, NY 10021 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 20, 2004. Contributed by Kathryn V. Anderson, February 8, 2005 Many aspects of the genetic control of mammalian embryogenesis proaches in other laboratories that focused on later stages of cannot be extrapolated from other animals. Taking a forward embryonic and fetal development in the mouse have also genetic approach, we have induced recessive mutations by treat- identified novel mutations successfully (9–11). With the avail- ment of mice with ethylnitrosourea and have identified 43 muta- ability of the mouse genome sequence, it has become straight- tions that affect early morphogenesis and patterning, including 38 forward to identify the genes responsible for the mutant genes that have not been studied previously. The molecular lesions phenotypes. responsible for 14 mutations were identified, including mutations Here, we describe the identification of 43 recessive mutations in nine genes that had not been characterized previously. Some that map to single loci and cause clear morphological abnor- mutations affect vertebrate-specific components of conserved sig- malities in the midgestation mouse embryo. Most of the muta- naling pathways; for example, at least five mutations affect pre- tions affect previously uncharacterized genes that control a viously uncharacterized regulators of the Sonic hedgehog (Shh) variety of processes, including the morphogenetic movements of pathway. Approximately half of all of the mutations affect the gastrulation and early neural patterning. Developmental mech- initial establishment of the body plan, and several of these produce anisms are conserved among mammals, and several mutations phenotypes that have not been described previously. A large described here will help define the developmental basis of fraction of the genes identified affect cell migration, cellular inherited human syndromes. organization, and cell structure. The findings indicate that pheno- type-based genetic screens provide a direct and unbiased method Materials and Methods to identify essential regulators of mammalian development. Identification of Mutants with Abnormal Morphology. We used the ENU mutagenesis protocol and crossing scheme defined previ- ethylnitrosourea ͉ hedgehog ͉ intraflagellar transport ͉ mesoderm ͉ ously, in which C57BL͞6J (B6) males were treated with three neural tube closure doses of 100 ␮g of ENU per gram of body weight, and all outcrosses were to C3HeB͞FeJ (C3H) females (8). For each F1 ammalian embryogenesis differs fundamentally from the founder male, five to eight G3 litters of embryos potentially Mdevelopment of other animal models. Localized maternal homozygous for newly induced mutations were analyzed. Can- components are not essential for axis specification in mammals didate mutants were identified when more than two embryos of (1), whereas the establishment of the body axes in Drosophila, similar, distinctive abnormal morphology were identified in a GENETICS Caenorhabditis elegans, and zebrafish relies on localization of line. Additional G2 ϫ F1 or G2 ϫ G2 crosses were analyzed to maternal determinants (2–4). Mammalian development has a confirm the presence of a genetic variant. We examined the unique requirement for complex interactions between embry- morphology of embryonic day 9.5 (e 9.5) G embryos from 380 onic and uterine tissues, and the first cell fate decision in the 3 lines. Based on the specific locus test data (7), there should be mouse embryo is the choice between embryonic and extraem- Ն30 newly induced gene-inactivating mutations in each line. By bryonic lineages (5). After implantation, germ layer organiza- using this estimate, the lines studied here would represent tion and tissue specification in the mouse embryo depend on Ϸ10,000 gene-inactivating mutations, affecting Ϸ25–30% of the coupled morphogenetic movements and intercellular signals, processes not paralleled in invertebrate embryos. A standard genes in the genome. approach to study the genetic control of mouse embryogenesis has been to inactivate evolutionarily conserved genes by targeted Linkage Mapping. All lines that produced reliable heritable phe- mutagenesis; this approach may overlook components that are of notypes were inherited as simple recessive Mendelian traits with particular importance in mammals. In contrast, phenotype- based screens in the mouse have the potential to identify the Abbreviations: ENU, ethylnitrosourea; SSLP, simple sequence length polymorphism; en, molecules that control mammalian-specific events. embryonic day n; Smo, Smoothened; bnb, bent body; Hh, Hedgehog; IFT, intraflagellar Phenotype-based screens depend on the ability to induce a transport; lzme, lazy mesoderm. large number of random mutations in germ cells and on rapid See accompanying Biography on page 5910. identification of mutants of interest. It has been known for 25 †Present address: Department of Molecular Biology, Princeton University Lewis Thomas years that ethylnitrosourea (ENU) is an extremely potent mu- Laboratory, Washington Road, LTL110, Princeton, NJ 08546. tagen in the mouse (6, 7); and a screen of the progeny of only 700 ‡Present address: Department of Human Genetics, Emory University School of Medicine, F1 progeny of ENU-treated animals should yield an average of Whitehead Research Building, 615 Michael Street, Suite 301, Atlanta, GA 30322. one allele of each gene in the genome. We previously described **Present address: Department of Cell and Developmental Biology, Weill Cornell Medical a pilot phenotype-based screen to identify recessive mutations College, 1300 York Avenue, New York, NY 10021. that produce easily visible disruptions in the morphology of the ††To whom correspondence should be addressed. E-mail: [email protected]. midgestation mouse embryo (8). Similar phenotype-based ap- © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0501071102 PNAS ͉ April 26, 2005 ͉ vol. 102 ͉ no. 17 ͉ 5913–5919 Downloaded by guest on October 1, 2021 Table 1. Alleles of previously characterized genes Mutant Phenotype Gene Mutation Nature of allele bent body (bnb) Dorsalized spinal cord, left-right randomization, die at e9.0 Smo (19) Splice site Null 2A Dorsalized spinal cord, left-right randomization, die at e9.0 Kif3a Not determined Null flexo (fxo) Abnormal diencephalon, no floor plate in caudal spinal Polaris (13) Splice site Hypomorph cord, left-right randomization, polydactyly, die at e13–14 M1un Arrested unturned, variable cardia bifida; arrest at e9.0 Pten Missense (M134K) Hypomorph DD1 Caudal paraxial mesoderm replaced by neural tubes Tbx6 Splice site Null complete or nearly complete penetrance. To map the mutation in a single line. Because Ͼ10% of lines contained at least one responsible for the phenotype, an initial genome scan panel used mutation of interest, this was approximately the number ex- DNA from adult parents of mutant litters identified in test pected in a sample of 43 mutations. In four cases, the phenotypes crosses (obligate carriers). A panel of 40 MIT simple sequence were different and segregated independently so that it was clear length polymorphism (SSLP) markers (http:͞͞mouse.ski.mskcc. that more than one mutation was present in the line. In one case, org) analyzed in 20 carriers defined linkage of the mutation to two different mutations affecting neural patterning arose on the a single segment of the C57BL͞6J genome for a majority of same chromosome but could be separated by recombination. mutations. In the remaining cases, the initial genome scan ruled out most genomic segments, and linkage was established with Map-Based Gene Identification. For lines with interesting pheno- analysis of additional markers and͞or additional carriers and types, we bred animals to generate embryos for phenotypic mutant embryos. Forty-three mutations were mapped to single analysis. These additional carriers and embryos provided enough genomic regions. In addition, four lines that produced interesting opportunities for recombination so that the gene could be phenotypes did not map to single loci; these and other lines were mapped to a small genomic interval. The MIT SSLP markers preserved as frozen sperm. could be used to map most genes to 1- to 2-cM intervals, but For most genomic segments, mutations could be mapped to those intervals generally included too many candidate genes to Ͻ5 cM by using MIT markers and a small number of additional evaluate. Generation of previously uncharacterized SSLP mark- carriers and mutant embryos. Higher resolution mapping re- ers and analysis of Ϸ200 litters of embryos and their parents quired the generation of additional polymorphic