The development of the vertebrate central nervous system (CNS) is orchestrated by complex genetic programs and cell extrinsic signals that govern the differentiation of neural progenitors into the rich variety of neuronal subtypes seen in the adult. While the molecular mechanisms that regulate general aspects of early neurogenesis have begun to be elucidated, the mechanisms controlling lineage-specific neuronal fate determination and differentiation remain largely elusive. In particular, the molecular-genetic programs that regulate the complex neuronal heterogeneity of the mammalian neocortex are only beginning to be discerned. Corticospinal motor neurons (CSMN), located among many other neuron types in layer V of the neocortex, are a prototypical and clinically important lineage for elucidating molecular regulation of cortical neuron subtype development. Recently, members of our laboratory developed approaches to purify CSMN (and other projection neuron lineages) from other neuronal and glial subtypes for microarray analysis, revealing a combinatorial program of lineage-specific molecular-genetic controls during CSMN specification and differentiation. These studies identified a number of candidate genes that appear to be critical regulators (each of the first four studied are newly identified central controls over CSMN development). In order to determine developmental functions and create a more complete picture of how this prototypical cortical neuronal subtype develops, I propose to investigate selected candidate genes: 1. cs3 and cs4, two transcription factors previously uncharacterized in the forebrain, will be examined via analysis of loss- and gain-of-function phenotypes as well as functional interactions of the two proteins;2. A very focused set of related candidates will be examined via a systematic gain- and Ioss-of-function analysis using in vivo electroporation and retroviral transduction-mediated overexpression and RNA interference (RNAi) approaches. Together, these studies aim to characterize the functions, temporal courses, and combinatorial roles of key molecular controls over CSMN development, elucidating lineage specification in the CNS. Significant clinical implications to this work also exist. Projection neurons are selectively vulnerable to disease and injury;CSMN vulnerability is especially apparent in motor neuron degeneration associated with amyotrophic lateral sclerosis (ALS) and loss of motor function after spinal cord injury. Understanding the developmental mechanisms that build CSMN will be a key first step in potentially developing effective and long-lasting CSMN protection and repair in the CNS. These advancements might also lead to generalizable investigative and therapeutic approaches for other neuron subtypes vulnerable to disease and injury.