Santos J. Franco, Ph.D. - US grants

DESCRIPTION (provided by applicant): Disruption of the laminar architecture of the neocortex is associated with more than 25 human neurological disorders, including epilepsy, schizophrenia, autism and mental retardation. Cortical layers are established by the migration of neurons from proliferative zones into the developing cortical wall. Thus, knowledge of how neuronal migration is regulated is critical for elucidating the mechanisms by which layer formation is achieved, and will likely provide insights into the pathological changes associated with several neurological disorders. My long-term objective is to define the mechanisms that control development of the laminar structure of the cerebral cortex. As a first step, I propose here to study the mechanisms by which reelin controls the formation of cortical cell layers. The central hypothesis of my proposal is that reelin targets distinct cellular functions in RGCs and migrating neurons that ultimately control cortical lamination. To test this hypothesis, the following specific alms will be pursued: Aim 1: Determine the mechanisms by which reelin affects RGC behavior. The proposed methods for achieving this goal are: (i) develop based on CRE/LOX recombination, siRNA expression and in utero gene transfer to perturb reelin signaling in RGCs without affecting signaling to neurons, (ii) use real-time imaging to determine the cell-autonomous defects in RGC process outgrowth and attachment that result from inactivation of reelin signaling, (iii) determine the extent to which defects in RGCs secondarily affect migration of cortical neurons, (iv) target cell-surface receptors implicated in reelin signaling to determine their roles in RGC function. Aim 2: Determine the mechanism by which reelin controls the migratory behavior of cortical neurons. The proposed methods for achieving this goal are: (i) use strategies similar to those described in Aim 1 to selectively inactivate reelin signaling in migrating neurons and study effects on their behavior, such as motility, development of polarity and somal translocation, (ii) developed mutant mouse lines suitable for selective genetic perturbation of reelin signaling in early- or late-born neurons, (iii) test the extent to which inactivation of reelin signaling during different modes of migration affects cortical lamination. Relevance: Abnormal development of the cerebral cortex causes more than 25 different human neurological syndromes that are characterized by significant clinical symptoms, including epilepsy, autism, schizophrenia and mental retardation. Therefore, understanding how the cerebral cortex is formed during development of the brain is expected to provide important information on the pathology of these diseases.

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.