Manipulating the Mouse Genome: llmplications for Neurobiology Review

I. Rossant Division of Molecular and Developmental Biology Samuel Lunenfeld Research Institute Mount Sinai Hospital and Department of Medical Genetics [Jniversity of Toronto Toronto, Ontario, M5G IX5 Canada introduction llnravelling the genetic control of the development of I he central nervous system represents one of the most challenging tasks facing mammalian developmental biologists today. Several different phases of develop- ment can be defined. The first event is neural induc- tion, segregating the neural plate from the rest of the embryonic ectoderm (Spemann, 1938; Gurdon, 1987). As the neural tube elongates, it appears that spatial domains are established that will later define specific regions of the brain and spinal cord (Sharpe et al., l987; Davis and Joyner, 1988; Gaunt et al., 1988; Duboule and DoIlk, 1989; Graham et al., 1989; Lums- den and Keynes, 1989; Wilkinson et al., 1989). Neuro- genesis itself begins shortly after and continues for varying lengths of time depending on the region of the nervous system (Hartenstein, 1989). Separation of the neural and glial lineages also occurs during this process, although the exact time of separation is not always clear. Finally, neural proliferation ceases, neu- rons establish their specific axonal connections, and Ihe final differentiated phenotype of the cells be- comes apparent. For a full understanding of this pro- cess, we need to know how cell lineage decisions are controlled genetically and how genes modulate neu- I’onal phenotype. A concerted attack on the cellular and molecular events of mammalian nervous system development requires many different approaches. Here I will restrict myself to those approaches that in- volve genetic manipulation of the mouse embryo. Genetic modification of the mouse embryo can in- volve both somatic tissues and the germline. Addition of novel genetic material to somatic tissues can be ,lchieved by introducing genetically marked cells to the embryo or by direct introduction of the genetic material into the cells of interest in the intact embryo. Modification of the germline can be produced by in- jection of DNA into the one-cell zygote, by retroviral infection of the early embryo, or by introduction of pluripotent embryonic stem (ES) cells into blastocysts. am going to consider two applications of these tech- niques that are particularly relevant to studies of the inervous system. I will discuss first the use of genetic marking for studying cell lineage in the nervous sys- tem and second the use of genetic manipulation to determine the function of specific genes in devel- opment. lineage Analysis Until recently the chief approach to lineage analysis in mouse development was to follow the fate of genet- ically marked cells in chimeras (Rossant, 1984). This approach has several limitations, some of which can now be overcome by application of two new tech- niques, retroviral cell marking (Price, 1987; Sanes, 1989) and genetic ablation (Bernstein and Breitman, 1989) or toxigenics (Evans, 1989). Chimera Analysis Genetic chimeras can be made during preimplanta- tion mouse development by either aggregation of cleavage stage embryos (Mintz, 1965; Tarkowski, 1965) or injection of cells into the blastocyst (Gardner, 1968). In either case, the end result is a mouse that consists of a fine mixture of cells of the two original genotypes. In theory, retrospective analysis of the genotype ratios in different cell types can provide some information on lineage relationships. For exam- ple, cells of related lineages should share the same genotype ratios and differ from cells of unrelated line- ages. Also, application of the binomial theorem or de- termination of the smallest contribution from one genotype to any given tissue should indicate the pro- genitor cell number for the tissue and allow estimation of the time of allocation of that lineage. In practice, calculations of this sort are fraught with difficulties, stemming from a variety of problems, some trivial, some more fundamental. One essentially trivial problem has been that, until recently, there were few good lineage markers to al- low detailed quantitation of the sort necessary for these calculations. lsozymes of ubiquitous enzymes like glucose phosphate isomerase (Chapman et al., 1971) can provide a rough idea of the overall genotype ratio in a complex tissue, but provide no information on the distribution of the genotypes in different cell layers or different regions. Ideally a lineage marker should be both ubiquitous and detectable in situ. A number of in situ markers have been applied to analy- sis of the nervous system in chimeras (Condamine et al., 1971; Dewey et al., 1976; Goldowitz and Mullen, 1982a), but none has proved of widespread applica- bility. Ubiquitous in situ nuclear markers are now available based on differences in repetitive DNA se- quences between different species or strains of mice. The first such marker system allowed cells from M. musculus to be distinguished from those of M. caroli in chimeras by in situ DNA-DNA hybridization to sat- ellite DNA sequences (Rossant et al., 1983). Essentially the same approach has now been applied to chimeras