Vertebrate Embryos depend on factors that are provided by the mother and are deposited into the egg or are supplied extraembryonically to the embryo for its normal development. Evidence from embryological manipulations and molecular embryology has shown that maternal factors are required for primordial germ cell development, mesoderm formation, establishment of the dorsal-ventral and animal- vegetal axes, and general cellular processes prior to the onset of zygotic transcription in amphibian and fish embryos. Recent work suggests that some of these maternal factors may reside in extraembryonic tissues is mammals. Little is known about the molecular nature of these maternal factors and the extent of their involvement in normal embryonic development. This study exploits the ability to perform large-scale genetic screens in the zebrafish, Danio rerio, to identify and characterize mutants of key genes specifically required in the mother for egg activation, fertilization, development of the germ line, and establishment of the body plan of the vertebrate embryo. Such large-scale maternal-effect mutant screens have not been previously performed in a vertebrate. This maternal- effect mutant screen will be performed through a three generation natural mating strategy to generate mothers homozygous for mutations in maternal-acting genes. Specifically, it is proposed to: 1.) perform a maternal- effect mutant screen of 2000 ENU mutagenized genomes for defects in primordial germ cell and embryonic body plan formation. 2.) Characterize mutant embryos or oocytes through fine morphological analysis, whole mount antibody stainings, in situ hybridizations, and other staining methods to determine if the mutants are defective in egg activation, fertilization, early cellular cleavages, DNA segregation, cell survival, or in cell fate specification of differentiation. This analysis will provide important information regarding the precise defects associated with loss of a particular maternal factor and possible molecular pathways that are defective in the mutants. 3.) Map a subset of maternal-effect mutations to chromosomal positions to facilitate propagation of the mutant strain and to identify the molecular nature of the maternal-effect gene by examining cloned genes mapping to the same genetic interval. Mutations are expected in genes involved in germ layer development, embryonic axis formation, anterior-posterior pattern formation, morphogenesis of the embryo, egg activation, fertilization, primordial germ cell development, and other critical developmental processes. These studies will be relevant to the nature of human sterilities, birth defects, and human inherited disorders.
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| Pelegri, Francisco; Mullins, Mary C (2004) Genetic screens for maternal-effect mutations. Methods Cell Biol 77:21-51 |
