Analysis of Differential Gene Expression by Boolean Selection: Identification of Transcripts Associated with the Formation and Reorganization of Neuronal Networks.

The quantitative comparison of gene expression represents one of the major approaches for the analysis of gene function. Recently, the discovery of unknown genes based only upon their expression pattern has become possible through the application of various differential cloning methods, including subtractive hybridization, differential display, and expressed sequence tag analysis. Due to numerous technical and procedural limitations, however, none of these technologies is currently capable of rapidly identifying differentially regulated transcripts based on multiple expression-related criteria. Therefore, an alternative approach was devised which allows for the sequential application of numerous selection criteria during the initial screening process, thereby enabling the rapid identification of transcripts with highly specific expression patterns. Because of its use of multiple positive and negative selection steps, this approach is called Boolean selection. One of the first applications of this new approach was the search for novel genes associated with the formation and reorganization of neuronal networks in the mammalian brain. An ideal model system for this analysis is the retinocollicular system of the rat, where numerous aspects of neurite outgrowth, axon guidance, synapse formation and reorganization, and regulation of neuronal survival can easily be studied. Boolean selection was applied for the identification of transcripts preferentially expressed in the superior colliculus during late prenatal development as well as after the loss of afferent input in the adult animal. An initial analysis of several of these selected transcripts revealed that all satisfied at least one selection criterion and that over half satisfied both, indicating the usefulness of Boolean selection for the rapid identification of differentially regulated genes. Although additional analysis by in situ hybridization will still be necessary to verify the extent of differential expression and to provide information on the cellular localization of the transcripts identified so far, it was possible with this preliminary screen to gain new insight into some of the molecular and cellular activities in the superior colliculus during periods associated with the formation and reorganization of synaptic connections. Some of the identified transcripts encode proteins associated with post-transcriptional protein synthesis and modification events, including ribophorin (an essential subunit of the protein glycosylation complex ligosaccharyltransferase), calnexin (a membrane-associated molecular chaperone of the endoplasmic reticulum), and translation initiation factor, which together suggest enhanced levels of protein production and secretion. Other transcipts include rSec6 (part of the protein complex involved in the docking and fusion of synaptic vesicles to the plasma membrane) and Fyn (a nonreceptor tyrosine kinase found at high concentrations in axonal growth cones and also known be involved in synaptic plasticity), Fas-associated protein factor (a recently identified molecule potentially involved in Fas-mediated signal transduction during program m ed cell death), and a novel member of the EOF superfamily of transmembrane growth factors (which presently includes the EGF/TGFa family and the neuregulins). Further analysis of these molecules may provide additional information about the signaling mechanisms and cell-cell interactions at work during the establishment and modification of neuronal networks.