Skeletal muscle provides a unique opportunity to investigate molecular genetic mechanisms that control specification of stem cell lineages within the cellular complexity of the vertebrate embryo. The proposed studies investigate developmental mechanisms that regulate the myogenic regulatory genes, MyfS and MyoD, which control the specification of skeletal muscle lineages from somite progenitors. Experiments focus specifically on the molecular mechanisms by which Sonic hedgehog (Shh) and Wnt signal molecules, produced by surrounding tissues, induce the transcriptional activation MyfS and MyoD in somitic progenitor cells, and thus initiate the process of myogenic cell specification. Complementary approaches have been developed to investigate these mechanisms in mouse and avian embryos. First, MyoD and MyfS transcriptional enhancers will be characterized using transgenic and transfection reporter gene assays, in combination with site-directed mutagenesis and nuclear factor footprinting, to map regulatory sequences and identify interacting transcription factors hypothesized to mediate Shh and Wnt induction of MyoD and MyfS.These studies undertake to distinguish whether the Gli and (3-catenin/LEFtranscription factors, which mediate Shh and Wnt signal transduction, interact directly with MyoD and MyfS enhancers to control their activation, or alternatively,whether these signals function upstream to regulate somite-specific transcription factors. Second, the developmental functions of a set of somite-activated regulatory genes, identified in differential cDNA cloning screens, will be investigated by antisense inhibition and misexpression analyses in avian embryos and by gene targeting and genetic analyses in the mouse embryo to determine their specific functions in MyoD and MyfS regulation during somite formation. These studies will focus on a family of two novel Sulfatases that were identified in our screen for somite-activated genes and are required for activation of MyoD in somites of avian embryos. These Sulfatases are hypothesized to desulfate proteoglycans in the extracellular matrix, thus controlling the localized activities of developmental signaling molecules essential for MyoD activation. Other novel somite-activated regulatory genes, identified in these screens, will be investigated to define their functions in the control of somite formation, Shh signal transduction, and MyoD and MyfS activation. The findings of these investigations are expected to lead to the discovery of novel developmental regulatory genes that control the specification of myogenic cells and other lineages in vertebrate embryos. Such knowledge will define fundamentalmechanisms that control of stem cell formation, and this information will likely lead to the development of new technologies to promote stem cell renewal in the repair of human tissues and organs damaged by disease and trauma.
