From Developmental Biology to Developmental Toxicology RUDI BALLINGa AND MARTIN HRABÉ DE ANGELIS Institute of Mammalian Genetics, GSF-Research Center for Environment and Health, 85758 Neuherberg, Germany
ABSTRACT: Progress derived from the human genome project will have tremendous impact on toxicology. Questions concerning genetic susceptibility or resistance to toxic compound exposure and the dissection of the molecular mechanisms involved will be at the forefront of future toxicological research. In recent years, it was recognized that many of the molecular control mechanisms of embryogenesis have been conserved during evolution. The relevance of these observations for toxicology and the application of genetic approaches using mouse mutants as a tool for functional genome analysis are discussed.
INTRODUCTION It is now 20 years since Christiane Nüsslein-Volhard and Eric Wieschaus published their paper about a large-scale mutagenesis screen in fruit flies.1 The authors described the isolation of a wide range of Drosophila mutants representing at least 15 loci with defects in the segmentation of the insect body pattern. It took less than 10 years to clone the genes responsible for most of these mutants and to develop a conceptual framework of how positional information is specified during insect embryogenesis. The biggest surprise from all of this work was the discovery that similar developmental control genes, with a high degree of DNA sequence conservation, could also be found in other organisms, including mice and humans. We now know that not only the DNA sequence, but also entire regulatory networks are conserved throughout the animal kingdom.
GENETIC APPROACHES TO DEVELOPMENTAL QUESTIONS Applying a genetic approach to a developmental biology problem turned out to be an incredibly powerful strategy. By first looking for mutants with a specific phenotype and then trying to find the responsible genes, one was not dependent on preconceived ideas of which pathway or molecular mechanism would be involved. Needless to say, this approach resulted in many surprises. Despite their large number, the Drosophila mutants could be grouped into a reasonable number of classes based on phenotypic criteria. These included gap, segmentation, segment polarity, and homeotic mutations. Other criteria were based on whether the affected genes act maternally or zygotically, or are transcription factors, secreted factors, ligands, or [email protected]
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ceptors.2 Elegant molecular embryological, cell biological, and biochemical experiments led to the recognition of a few major signal transduction pathways that seem to play a role in almost every process during embryogenesis.3–5 Segmentation of the insect embryo was thereby used as a first entry point into the general mechanisms of how cells become different from each other, how they proliferate or differentiate, and how they know where to do what and for how long.6,7 We now know, for example, that the “Sonic hedgehog signaling pathway” not only determines whether the surface of a Drosophila cuticle is covered by bristles or not. We also know that this pathway is essential for other aspects of Drosophila development, as well as for vertebrate organogenesis, such as eye, kidney, lung, heart, limb, and brain development.8–11 The famous statement from J. Monod, “what is true for E. coli is true for the elephant”, turned out to be equally true for developmental biologists 20 years later. However, it was not only the field of developmental biology that was revolutionized by the recognition of evolutionary conservation as the integrative element in biology. Cancer biologists now have plenty of examples demonstrating that developmental control genes can be synonymous with oncogenes or tumor suppressor genes.12–17 The same genes that are important for development of an organ are often involved in maintaining integrity and homeostasis of this organ during the entire life of an organism. By studying embryogenesis and organogenesis, we often get important insight into the pathogenesis of diseases in adults. On a molecular basis, this translates into the discovery that genes can play many roles during the life of an organism.18,19 A gene that is essential for gastrulation might also play a role in blood pressure regulation, memory, or wound repair. What does this all have to do with toxicology? Toxicologists try to explore which compounds at which concentration and through which mechanisms are toxic to humans and other organisms. Molecular biology has given us the tools to dissect the responses of a cell or an entire organism after exposure to a toxic compound in terms of the molecules involved. Toxicologists will therefore need to study the genes and proteins that are induced, repressed, or modified after toxic exposure. Developmental biology provides us with the concepts of how these genes are connected with each other and what their functional significance might be during the development of an organism. Signal transduction pathways recognized by toxicologists as responses after toxic exposure turn out to be the same as those that developmental biologists have identified as regulators of embryonic processes.20 Recognizing these similarities helps tremendously in the interpretation not only of the molecular data, but also of associated pathophysiological phenotypes. The relevance of developmental biology is particularly obvious in the field of teratology. It is known for quite some time that certain drugs or chemicals can induce phenocopies of congenital developmental defects. Drugs or toxic compound exposure can lead to the same phenotype as the effect of a mutation in a specific developmental control gene. One of the most convincing examples of the convergence of developmental biology and teratology is the pathogenesis of retinoic acid embryopathy. Retinoic acid has been identified as a teratogen in the 1980s.21 However, the molecular basis of how retinoic acid exerts its effects (i.e., when taken by pregnant women for the treatment of acne) remained unknown. Two independent observations solved this question. One of these was the discovery by the group of Eduardo Boncinelli that adding
BALLING & HRABÉ DE ANGELIS: DEVELOPMENTAL TOXICOLOGY
retinoic acid to human embryonic carcinoma cells cultured in vitro led to the induction of the expression of Hox genes.22,23 Which of the 38 Hox genes were turned on depended on the position within one of the four chromosomal Hox gene clusters. Genes located more 5′ in a cluster were turned on later than those located more 3′. The second important observation involved the production of transgenic mice that overexpressed the homeobox gene Hox 1.1, now called Hox A7.24,25 Ectopic expression through a ubiquitous chicken β-actin promoter resulted in a phenotype that was very reminiscent to that seen in retinoic acid embryopathy. It turned out that the application of retinoic acid during embryogenesis led to ectopic expression of Hox genes and thereby resulted in the same phenotype as when Hox genes were expressed at the wrong time or in the wrong place. The fields of developmental biology and teratology thus met at the level of gene regulation and gene deregulation. In the meantime, we know of many more examples in which developmental pathways turn out to be the target of a teratogen. Shh mutations cause holoprosencephaly in humans26 and so does exposure to cholesterol inhibitors.27,28 Homeotic mutations are induced in mice after exposure to valproic acid, which is a commonly used anticonvulsant.29 A very similar phenotype can be observed in mice with loss-offunction mutations in Hox genes or retinoic acid receptor genes.30,31 One of the exciting challenges for the future is the question of how mutations in human genes, which alone do not lead to developmental defects, might lead to an increased risk of toxicological or teratological side effects when these carriers are exposed to toxicological compounds. The field of toxicogenetics and toxicogenomics will move into the center stage of tomorrow’s toxicology research. This, of course, triggers the question of whether the genetic tools that were so successful for developmental biology could also be applied to the field of toxicology.
PHENOTYPE-DRIVEN VERSUS GENOTYPE-DRIVEN MUTAGENESIS As described earlier, one of the most successful strategies for dissecting the mechanisms of embryogenesis in Drosophila was the use of large-scale mutagenesis screens. Thousands of Drosophila embryos derived from mutagenized flies were scored for abnormal segmentation phenotypes.1 Recently, this kind of large-scale phenotype-driven mutagenesis has also been applied to mice.32–35 The mouse has developed into the most important model organism to investigate the genetics and pathogenesis of human disease.36 Mice are 2000- to 3000-fold lighter than humans. Their generation time is 10 weeks and, with a litter size of 5–10, there is no other mammal that can be kept and studied as cost-efficiently as the mouse. Mouse embryonic stem cell technology and homologous recombination have opened the possibility to produce mutants for any gene that is cloned. In a few years, the entire human and mouse genome will be sequenced, making this approach even more powerful and efficient. The majority of mutants that are produced by gene targeting will be insertional mutations that interrupt gene function. In most cases, these mutations will be null alleles. Complementary to such a gene-driven approach is a phenotype-driven approach, in which a gene is inactivated and then the resulting mice are analyzed for the phenotypic consequences. Mutants are produced by chemical mutagenesis or
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other means and mice with a desired phenotype are isolated from a large number of mutagenized animals. In these phenotype-oriented screens, there is no need to have any knowledge about the underlying genes or molecular mechanisms. Once a mutant has been found, genomic strategies, such as candidate or positional cloning, will allow the identification of the genes underlying the phenotype of interest. While this can take some time, the researcher starts from a phenotype in which he has a strong interest. Gene knockouts often do not result in a detectable phenotype or give rise to phenotypes that lie outside the expertise of the researcher. Furthermore, many abnormalities of knockout mice are missed due to inappropriate phenotypic characterization of the mutants. Having access to a large collection of mutant mice with specific inherited abnormalities would be an enormous tool not only as a model to study the pathogenesis of human diseases, but also for the analysis of basic biological mechanisms.
ENU MUTAGENESIS One of the strategies to produce a large number of mouse mutants is the use of chemical mutagenesis. ENU is an alkylating reagent and currently the most powerful mutagen in mice.37 ENU induces mainly A-T substitutions and, if given at the most efficient dose, a mutation frequency of more than 1 in 1000 can be achieved.18,38–40 Male mice are injected with ENU and then mated to normal wild-type females. The first generation of offspring, the F1 animals, are then analyzed for dominant traits or bred further to identify mutations that lead to recessive phenotypes. Large numbers of mice can be easily scored for dominant visible abnormalities. A genome-wide scan for recessive phenotypes is much more labor-intensive, but other strategies (e.g., region-specific screens with mice that already carry a deletion) provide interesting alternatives.41 Currently, two large-scale ENU mutagenesis screens are being carried out32,34—one at the GSF Research Center in Munich and the other at the MRC Mammalian Genetics Unit in Harwell (United Kingdom). Additional screens are in preparation in Japan, the United States, and Australia. In the meantime, the labs in Harwell and the GSF have produced about 200 new mouse mutants each, providing a rich source of mutants and a wide range of phenotypes. Whereas the screen in Harwell focuses on the isolation of neurodegenerative and behavior mutants, the GSF screen is primarily targeted towards the isolation of congenital malformations, biochemical alterations of clinical relevance, and immunological and hematological defects. Unlike gene targeting or insertional mutagenesis, mutations produced by ENU are not molecularly tagged. In order to identify the genes affected, positional or candidate cloning is required. A prerequisite for this is the determination of the chromosomal localization of the mutation. This is achieved by backcross strategies and genome-wide microsatellite genotyping. The availability of the complete mouse genome sequence will have an enormous impact on the efficiency of these cloning efforts. Currently, major work is under way to develop necessary databases and cryoconservation archives for making the ENU mutagenesis–derived mutants available to the scientific community.
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THE USE OF ENU MUTAGENESIS IN TOXICOLOGY The phenotypic assays employed so far in the ongoing ENU mutagenesis projects are to a large degree influenced by the interests and capabilities of the labs involved. However, one of the important messages that has come out of these projects is that, for almost any phenotype that can be scored with a robust, cost-efficient, and reliable assay, mutants can be isolated. There are many assays and endpoints that are of high interest to toxicologists. Increased or decreased susceptibility and resistance to toxic compounds are among these. However, it is also the dissection of pharmacological mechanisms that is attracting increasing interest. Mutants with abnormal absorption, distribution, metabolism, or excretion (ADME) of drugs or toxic chemicals can provide important insight into the molecular mechanisms of individual drug efficacy or toxicity. Mouse ADME mutants isolated from ENU screens could be a great tool for human toxicogenetics and pharmacogenetics. Currently, the mouse is not used as intensively in toxicological research as it should. The rat, mainly for historical reasons, is still the major animal in toxicological risk assessment. Given that pharmacological and toxicological risk evaluation has to be based on the most updated knowledge, it is a question of time before the mouse takes over a major role in toxicology during the process of drug development. Although the rat will continue to be very important for specific questions, the advantage of the genetics of the mouse needs to be taken into consideration for future toxicological research.
OUTLOOK Toxicology of the new millennium will profit tremendously from the progress currently made in genomics and proteomics.42 With the availability of the complete human and mouse genome sequences within the next few years, DNA array technology will soon become a routine procedure in evaluating xenobiotic and drug responses.43–45 Through functional genomics, the role of individual genes as well as the interaction of many genes within the genome will be studied in toxicology and pharmacology. Individual predisposition as opposed to the responses at the population level will move to the forefront of toxicology research.46–48 Pretty soon, we will apply tools of complex system analysis to describe the consequences of toxic exposure to cells and organisms. Multigenic and multifactorial traits, genetic predisposition, global and genome-wide expression changes, and the integration of structureactivity relationships will all become integral components of toxicological hazard identification and risk assessment. A systematic production of new mouse mutants will facilitate to make the necessary rational and scientifically based decisions in future toxicological research. REFERENCES 1. NÜSSLEIN-VOLHARD, C. & E. WIESCHAUS. 1980. Mutations affecting segment number and polarity in Drosophila. Nature 287: 795–801. 2. PICK, L. 1998. Segmentation: painting stripes from flies to vertebrates. Dev. Genet. 23: 1–10.
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