Barbara J. Wold
Cell FateIn the Wold group we are interested in the problem of how mammalian cell fates are specified and executed. A major challenge is to understand at molecular and cellular levels the series of developmental decisions that lead from multipotential, precursor cells to their specialized, differentiated products. We use the mouse as our primary experimental animal and the specific lineages we study are mesodermal; they ultimately produce skeletal muscle, cardiac muscle, and several non-muscle somitic derivatives. Skeletal myogenesis is governed by both positive- and negative-acting regulatory factors. In this process, coordination of cell cycle regulation with regulation of differentiation is a hallmark of the system. A family of four closely related, positive-acting transcription factors appear central to muscle development. They are MyoD, myogenin, myf-5 and MRF4/herculin. Upon transfection into many types of non-muscle cells, each of these has the capacity to drive recipient cells into and through the myogenic pathway. Such transformants, under appropriate culture conditions, differentiate into skeletal myocyte-like cells that express a complex battery of muscle-specific proteins. Given their extraordinary power to drive or even redirect a cell fate decision, one experimental goal is to understand how MyoD family regulators function. Projects byXin Yu, Ardem Patapoutian, Jeong Yoon, Roger Wagner, and Dee Cornelison focus on different aspects of this problem, emphasizing new approaches to study their activities in in vivo settings. At the level of the whole organism, this includes gene disruption and gene transplacement by targeted homologous recombination to study the phenotypes of null mutations in muscle regulators, controlled ectopic expression in transgenic animals, the development and characterization of cell cultures and lines from early embryonic muscle precursors, and experiments to trace the fates of cells that have at an earlier time in development expressed key regulators. At the molecular level, this involves application of a novel strategy for probing the MyoD regulatory network (forced dimer polyproteins), and further development and application of in vivo footprinting to help understand how protein:DNA complex formation is regulated and related to gene activity in the cell.
Negative-acting regulators of muscle differentiation.At the cellular and molecular levels, it is clear that negative regulators of skeletal myogenesis are probably just as important in properly regulating the outcome as are the positive MyoD and MEF2 family regulators. The interaction between the two sets of regulators is of particular interest to us. Negative regulators of skeletal muscle differentiation that we find especially interesting and relevant are those that are expressed in multipotential mesodermal precursors or in proliferating muscle precursors (myoblasts), and it is postulated that some of these are important for specifying their precursor status and for maintaining precursor cells in an undifferentiated state. Others also appear to have a role in driving proliferation of the precursor compartments at the same time that they mitigate against execution of the muscle differentiation program. Regulators in these classes include twist, myc, Max/myn, Id, c-jun, and protein kinase C. Sonya Zabludoff Palmer, Kyuson Yun, and Marie Csete are studying different aspects of this problem.
Cell cycle progression and its roles in developmentA major problem in development is controlling cell proliferation and coordinating it with cell fate decisions so that the proper number of cells of each type are ultimately provided. Moreover, in many cell types the function of the final differentiated cell depends on its cell-cycle status. For skeletal muscle, differentiation is tightly linked with entry into a G0-arrested state. Work by Sonya Zabludoff, Marie Csete and Susan Wang is directed at understanding regulation and execution of the entry into the G0 state and the role, played by that c-myc, G1 cyclins, their cogante cdks, and their recently discovered inhibitory proteins play in directing or inhibitine that progression. A new and related thrust is our interest in myogenic satellite cells and understanding their lineal origin, cell cycle regulation and regulation of their myogenic status. Dawn Cornelison and Inna Gitelman are studying the functions and expression of cdk/cyclin inhibitory proteins in the context of mouse development, including p16/MTS1, p15/MTS2, p27/KIP1, p57 and p21.
This is the inevitable question when an set of regulators is discovered. In this case, the specific problem is to learn what turns on and then maintains expression of MyoD family members during development. Jeong Yoon is studying this problem with special attention to MRF4 and myf5. Studies of other regulators that appear important in the mesodermal lineages prior to expression of MyoD family members or in non-myotomal portions of somites focus on mouse twist, previously cloned and characterized in the laboratory. Kyuson Yun and Shuling Wang have looked at different aspects of its activity.