The focus of this amended application is cell specification and pattern formation in the mammalian musculoskeletal system. The proposed experiments rely heavily on the use of cutting edge and developing technologies, the recombinase mediated molecular lineage tracing (RBLT) and magnetic resonance imaging (MRI) for cellular lineage tracing. The third technology that is more routine is the use of homologous recombination to inactivate genes. The investigators will apply the MRI based lineage tracing to analyze development of craniofacial muscle and skeleton. The rationale for this is that such an atlas of development will prove to be useful in analyzing the impact of mutations on these structures. RBLT is a complementary technology that will allow tracing the fate of individual cells during development. This technology will interface with the experiments dealing with myogenic regulators. Strains of mice will be developed with alterations in myogenic regulatory genes. In addition, the fate of those cells that express myogenic regulators early in development will be traced. There have been several changes in the proposal since the original application was considered by the Review Committee in November 1993. First, Project 4 (the original application consisted of four projects and one Core unit), which was viewed by the Committee to be not closely tied in with the theme of the program project, has been deleted. Second, for Project 3 of the original application, one major criticism was the lack of detail and the potential lack of resolution of the method used. The investigators have included new preliminary data and acknowledged that more conventional methods could be employed if needed. Finally, the issue of the centralization of the Caltech transgenic mouse facility has been clarified. Both Drs. Wold and Anderson have a direct role in establishing this facility. They have interacted effectively in the past, although this interaction has not been formalized by joint publications. P01AR426710001 Description (adapted from the applicant's abstract): Four closely related transcription factors, MyoD, myogenin, myf5 and MRF4/herculin/myf6 are thought to function as major, positive acting regulators of skeletal myogenesis. The current view of their function in muscle determination and differentiation came initially from studies in cell culture models and from the observation that their expression is restricted to skeletal muscle and its immediate progenitors. This is presently being both supported and substantially modified by the ongoing analysis of mice in which myogenin, MyoD or myf-5 have been disrupted. A successful disruption of MRF-4 has not yet been achieved and published. This proposal will focus on two major questions concerning mammalian myogenic lineages: What are the functions of MRF-4 during development, and what roles do different MyoD family members play in restricting cells to a myogenic lineage? To address these questions, the investigators propose to construct and study strains of mice in which MRF-4 has been disrupted by the standard ES cell mediated design. Previously, when they cloned and characterized mouse MRF-4, they found that several properties distinguish it from the other three MyoD family members. Chief among these is the fact that MRF4 is by far the quantitatively dominant MyoD family member expressed in muscle of adult mice. This led the investigators to propose that MRF-4 plays the key role in maintaining muscle phenotype, and perhaps in mediating responses to activity level. The knockout will permit them to test directly this hypothesis and others concerning phenotypic interactions among different MyoD family members, autoregulation and cross-regulation in the HLH network and possible roles in specifying subsets of the skeletal lineage. As part of the Program Project, the investigators propose to help develop recombination based lineage tracing (Projects 1 and 2) and then use it to address several distinct questions about myogenic lineages and the roles played by MyoD family members in their specification. This novel analysis will permit them to examine the origin and determination of lineages leading to satellite cells and to the appendicular musculature. Each of these myogenic settings offers different features that will balance two goals of the Program. One is to probe the biological significance of MyoD family expression, whether transient or continuous, in a given lineage over a specific developmental time window. The second goal is to provide a well-controlled and experimentally accessible series of tests for Recombinase mediated lineage tracing in the mouse.
