The goal of this project is to develop a new molecular method, termed recombinase-based lineage tracing"""""""" (RBLT), to provide a fate map of embryonic precursor cells that express the MASH-1 gene, a nuclear regulatory gene of the basic helix-loop-helix (HLH) family. Recombinase mediated lineage tracing provides a means to mark the mitotic progeny by DNA rearrangement driven by a site-specific recombinase (Cre). A primary advantage of this approach is the access to neural precursor cell populations that would likely be inaccessible to dye labeling or retroviral marking methods. Since the review of the original application, several significant advances have been made. First, the investigators demonstrated that Cre-mediated recombinations of a lacZ reporter gene are feasible for fate mapping experiments, shown by crossing activator mice expressing a CMV-Cre construct to transgenic animals harboring a reporter gene, chick actin XSTOPX lacZ. The offspring generated showed no developmental defects, thus offsetting a concern raised in the previous review. Second, the investigators will utilize the green fluorescent protein (GFP) as an alternative to alpha-galactosidase. Finally, a recent publication describing a null mutation in the MASH-1 gene demonstrated an essential role for MASH-1 in the development of several classes of neurons, including the sympathoadrenal lineage and olfactory neurons. The Cre-recombinase methodology proposed should facilitate studies on other neural lineages that express MASH-1, e.g., progenitor cells in the forebrain.
