Analysis of mouse embryonic patterning and morphogenesis by forward genetics

Abstract

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.ethylnitrosourea ͉ hedgehog ͉ intraflagellar transport ͉ mesoderm ͉ neural tube closure M ammalian embryogenesis differs fundamentally from the development of other animal models. Localized maternal components are not essential for axis specification in mammals (1), whereas the establishment of the body axes in Drosophila, Caenorhabditis elegans, and zebrafish relies on localization of maternal determinants (2-4). Mammalian development has a unique requirement for complex interactions between embryonic and uterine tissues, and the first cell fate decision in the mouse embryo is the choice between embryonic and extraembryonic lineages (5). After implantation, germ layer organization and tissue specification in the mouse embryo depend on coupled morphogenetic movements and intercellular signals, processes not paralleled in invertebrate embryos. A standard approach to study the genetic control of mouse embryogenesis has been to inactivate evolutionarily conserved genes by targeted mutagenesis; this approach may overlook components that are of particular importance in mammals. In contrast, phenotypebased screens in the mouse have the potential to identify the molecules that control mammalian-specific events.Phenotype-based screens depend on the ability to induce a large number of random mutations in germ cells and on rapid identification of mutants of interest. It has been known for 25 years that ethylnitrosourea (ENU) is an extremely potent mutagen in the mouse (6, 7); and a screen of the progeny of only 700 F 1 progeny of ENU-treated animals should yield an average of one allele of each gene in the genome. We previously described a pilot phenotype-based screen to identify recessive mutations that produce easily visible disruptions in the morphology of the midgestation mouse embryo (8). Similar phenotype-based approaches in other laboratories that focused on later stages of embryonic and fet...

Tissue-specific roles of Axin2 in the inhibition and activation of Wnt signaling in the mouse embryo

“…The canopus (canp) mutation was identified in a genomewide N-ethyl Nnitrosourea (ENU)-mutagenesis screen for mouse developmental mutants (20), on the basis of the abnormal morphology of mutant homozygotes. Most homozygous canp embryos (75-80%) arrested at midgestation with abnormal hearts and slightly shortened tails ( Fig.…”

Mouse Kif7/Costal2 is a cilia-associated protein that regulates Sonic hedgehog signaling

“…ENU mutagenesis and the genetic screen were carried out as described in ref. 48, except that mutagenized C57BL6/J males and F1 males were mated with females homozygous for an HB9-eGFP reporter in the FVB genetic background, made by using a previously described construct (24) (Fig. S1 A).…”

Mouse mutagenesis identifies novel roles for left–right patterning genes in pulmonary, craniofacial, ocular, and limb development

“…Ethylnitrosourea (ENU) is a highly efficient mutagen in mouse, with specific mutation rates between 1 in 1,000 and 1 in 700 reported (Russell et al, 1979;Hitotsumachi et al, 1985). Both genome wide (Hrabe de Angelis et al, 2000;Nolan et al, 2000;Herron et al, 2002;Garcia-Garcia et al, 2005) and more directed regionspecific genetic screens (Shedlovsky et al, 1988;Rinchik and Carpenter, 1999;Kile et al, 2003;Bogani et al, 2005) have resulted in the identification of a large number of novel mutant alleles. However, dominant screens examining over 40,000 pedigrees (Hrabe de Angelis et al, 2000;Nolan et al, 2000) failed to identify mutations affecting L-R determination.…”