PROJECT SUMMARY The neocortex is crucial for execution of our higher order brain functions such as cognition, consciousness, perception and motor control. The complex neural circuits that underlie these functions are built from many different types of neurons and glia during brain development. How this cell type diversity is achieved from a common pool of neural progenitors in the developing forebrain is a major research focus, but there are still many fundamental gaps in our knowledge of this process. In particular, the molecular mechanisms that control glial cell fate specification and generation from neocortical progenitors are largely unexplored. The long-term goal of this project is to understand the mechanisms underlying cell type diversity and specification in the cerebral cortex and to use this knowledge for therapeutic purposes in the diseased brain. The objective of this proposal is to elucidate the mechanisms underlying oligodendrocyte specification and subtype diversity. Oligodendrocytes are essential for normal brain development and function, and their importance is underscored in diseases in which they are disrupted, including multiple sclerosis and leukodystrophies. Similar to neurons, recent studies have started to uncover diversity within the oligodendrocyte lineage that likely reflects their multiple functions in the neocortical circuitry. The early developmental origins of this oligodendrocyte diversity are not known. Preliminary data produced in the applicants' laboratory indicates that 1) oligodendrocyte lineage specification from neural progenitors begins early in neocortical development, before neurogenesis is complete; 2) Sonic hedgehog signaling to progenitors in the embryonic dorsal forebrain is critical for generating neocortical oligodendrocytes; and 3) heterogeneity within the neocortical oligodendrocyte lineage depends on precise regulation of Sonic hedgehog signaling levels. Based on these data, the central hypothesis is that embryonic Shh signaling restricts a subset of neocortical progenitors to oligodendrocyte identities, and differing levels of Shh signaling further specifies subtype fate within the oligodendrocyte lineage. This hypothesis will be tested by pursuing two specific aims using in vivo techniques in mice: 1) Under the first aim, daughter cells belonging to the dorsal Ascl1 lineage will be identified by genetic fate-mapping and in vivo clonal analysis, to test the hypothesis that Ascl1+ neocortical progenitors are oligodendrocyte-fate restricted; 2) Under the second aim, in vivo clonal analyses in combination with dose- controlled loss-of-function approaches will determine whether precise levels of Shh signaling control the ratio of different subtypes of oligodendrocyte-lineage cells. The proposed research is significant because it is expected to provide a better fundamental understanding of the molecular mechanisms underlying oligodendrocyte specification, and it is the first step toward new advances in deriving specific subtypes of oligodendrocytes from stem cells for therapeutic transplantation to combat demyelinating disorders.