FLP-MEDIATED CELL LINEAGE ANALYSIS IN THE EMBRYO

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The overall objectives of this project are to develop a binary recombination-based system to fate map and manipulate somatic lineages in the mouse embryo, and to utilize this new tool to reveal mechanisms underlying establishment of neural crest cell lineages. A central issue in mammalian development -understanding how lineage and environment interact to determine phenotype - has been limited by the inability to follow the fate of specific cells in situ and to observe the distribution of their progeny in the embryo throughout gestation. Such precise lineage mapping is prerequisite to understanding developmental determinants of cell proliferation, differentiation, and migration. Knowledge of these mechanisms is fundamental to understanding the origins of congenital malformations. We have engineered an in situ lineage marking system in transgenic mice that should mark specific populations of cells in a heritable, cell autonomous, non-diluting fashion during embryonic development. Additionally, this system can be used in conjunction with homologous recombination to perform lineage/tissue-specific in vivo mutagenesis. We have exploited an excisional recombination system found in yeast for these purposes: the recombinase FLP catalyzes recombination between direct repeats of FLP recombination targets (FRTs) excising the intervening DNA. Since initiating this project in 1993 we have: (1) constructed a modular set of FLP and universal target (FRT-disrupted lacZ) vectors; (2) demonstrated efficient FLP-activation of the target transgene in embryonic stem (ES) cells; (3) generated separate FLP and target (FRT-disrupted lacZ) transgenic mouse lines; and (4) demonstrated FLP-mediated recombination in embryos from a FLPxtarget cross. We are now in the unique position to fully characterize this system in vivo as a tool for marking cell lineages and for directed modifications of the mouse genome. Because neural crest cells migrate extensively and differentiate to form a variety of cell types they are optimal to study how lineage and environment interact to determine cell fate. Derangements of neural crest cell development are implicated in numerous congenital malformations including neural tube, limb, cranial, enteric ganglion and cardiac defects, deafness, and thymic agenesis. A major unanswered question is when and how does this population of cells generate such phenotypic diversity. Toward the proposed aims, we have generated transgenic mice expressing FLP in the dorsal CNS as a means to activate lacZ in neural crest progenitors. We will: (1) utilize this marking system to map the murine neural crest; (2) identify similarities and differences between the murine map and that of the chick, the latter providing much of our current knowledge; (3) move from descriptions of cell fate to analysis of underlying mechanisms through lineage studies in mutant embryos. Mechanisms of pathogenesis associated with Splotch (Pax-3), lethal spotting (Is), and W (c-kit) phenotypes will give insight into the pathogenesis of parallel human syndromes (e.g. Waardenburg Syndrome and Hirschsprung's disease).