We have produced monoclonal antibodies (mAbs) that recognize candidate downstream targets of homeobox transcription factors. The antigen for one of the mAbs, FORSE-1, is the focus of this project. FORSE-1 binds the surfaces of proliferative neural precursor cells in the embryonic forebrain. It's pattern of labeling delineates a novel set of boundaries in the rodent telencephalon and diencephalon, displaying a partial overlap with the expression of the transcription factor BF- 1. We have identified the FORSE-1 epitope as the oligosaccharide Lewis x (Le x), and the antigens that carry this epitope in the brain as neutral glycolipids and the chondroitin sulfate proteoglycan, phosphacan. We propose to investigate the function of Le x in vivo and in vitro. (1) Migration in vivo We have initially focused on the perturbation of neuronal migration in vivo by injection of the FORSE-1 mAb, control mAbs and synthetic Le x oligosaccharide antagonists. Injection of the synthetic antagonists or FORSE-1-secreting hybridoma cells into the ventricle of E15.5 rat embryos severely disrupts the migration of BrdU-labeled neurons to cortical layers 5 and 6. Injection of hybridoma cells secreting the HNK-1 mAb, in contrast, has no effect on migration of these cells. Additional positive and negative control mAbs and oligosaccharides will be tested in this in vivo system. (2) Migration in vitro In order to elucidate further the mechanism by which migration is inhibited, both the oligosaccharide perturbants and the mAbs will be used in a slice culture system in which the migration of BrdU- or DiI-labeled neurons are followed (using time-lapse imaging in latter case). (3) Axon guidance The in vivo perturbation procedure will also be used to test a role for LeX in axon guidance. Cortical and retinal axon tracts in mAb- or oligosaccharide-injected embyros will be labeled with TAG-1 and NF mAbs, and with DiI. Size of tracts, trajectory of growth cones and degree of fasciculation will be measured. (4) Fuc-T knockouts We have obtained mice in which two of the five known fucosyltransferases (Fuc-Ts) that synthesize Le x have been disrupted by homologous recombination. The brains of these knockout embryos will be analyzed for FORSE-1 labeling and, if negative, for defects related to neuronal migration and axon outgrowth in vivo and in vitro. If the available mice are not deficient in brain Lex , we will collaborate on cloning and disrupting a brain-enriched form of Fuc-T.
