Although there has been enormous progress toward understanding the intercellular signals that organize the developing vertebrate limb, practically nothing is known about the lineage relationships between cells that give rise to the various limb tissues, or of the differential fates of cells which produce the organizing signals. Defining such lineage relationships is essential to understanding limb morphogenesis. Here, a twofold strategy is proposed. First, individual cells derived from the limb lateral plate mesoderm (LPM) will be traced, by labeling with a complex library of oligonucleotide-tagged retroviruses, to ask when individual cells and their descendents become committed to particular cell fates and spatial locations. The LPM gives rise to the limb skeleton, connective tissue, smooth muscle and dermis, but how and when these populations diverge is unknown. Second, the progeny of cells in the Zone of Polarizing Activity (ZPA) will be traced. ZPA cells express Sonic Hedgehog (Shh), a secreted signal that polarizes the limb along its A/P axis. Permanent, heritable LacZ expression will be generated in all descendents of the Shh-expressing ZPA using the Cre-recombinase system. Some cells in the posterior limb bud, a region that includes the ZPA, undergo apoptosis while others contribute to digit structures. Whether descendents of the ZPA can reprogram gene expression patterns and contribute to limb structures is unknown. If true, the identification of such dynamic shifts in Shh expression would have profound implications for the mechanism of limb patterning.
