MOLECULAR STUDIES OF FASCICLIN 2 IN NEURAL DEVELOPMENT

The proposed research is a molecular investigation of growth cone
pathfinding mediated by the fasciclin 2 (Fas2) protein, with the long-term
objective of understanding neural specificity by defining the biochemical
mechanisms for cell-cell recognition in the developing central nervous
system (CNS). The experimental approach is to study the expression and
function of Fas2 by application of molecular and genetic methods primarily
in the fruit fly Drosophila melanogaster, and cellular and anatomical
techniques mainly in the grasshopper, Schistocerca americana. Fasciclin 2
is a cell surface glycoprotein isolated from grasshopper which is very
similar in structure to vertebrate neural cell adhesion molecules (such as
N-CAM, L1, contactin and myelin-associated glycoprotein), having 5
immunoglobulin-type domains and 2 fibronectin-like regions. It is expressed
at first on a single longitudinal axon bundle in the CNS, and antibodies
against it disrupt the formation of this axon bundle by retarding or
blocking the initial fasciculation of its constituent axons. The specific
goals of my proposal are these: (a) isolation and characterization of Fas2
cDNA and protein from Drosophila melanogaster; (b) isolation of the gene fo
Fas2; and (c) isolation and characterization of Drosophila melanogaster
which lack a functional Fas2 gene; and (d) determination of the biochemical
mechanism of Fas2 action, including molecular structure-function studies on
Fas2 in transfected cells and germ-line transformed Drosophila embryos. Al
four specific aims will be approached initially by using the previously-
characterized grasshopper Fas2 cDNA to isolate new Fas2 cDNA and genomic DN
clones from Drosophila and grasshopper. Using standard techniques of
molecular biology, these DNA fragments will be altered in specific ways and
their function and expression tested after transfection into cell lines and
into the Drosophila germ line. The discovery of Fas2 provides a unique
opportunity to study a cell adhesion/recognition molecule using a potential
ly powerful combination of molecular genetic, cell biological and anatomica
methods in these model systems. The results from this project will help us
understand how related molecules such as human N-CAM may function in brain
development.