Dendritogenesis, axonogenesis, pathfinding, and target recognition are all affected in distinct ways when Xenopus retinal ganglion cells (RGCs) are transfected with constitutively active (ca), wild-type (wt), and dominant negative (dn) Rho-family GTPases in vivo. Dendritogenesis required Rac1 and Cdc42 activity. Moreover, ca-Rac1 caused dendrite hyperproliferation. Axonogenesis, in contrast, was inhibited by ca-Rac1. This phenotype was partially rescued by the coexpression of dn cyclindependent kinase (Cdk5), a proposed effector of Rac1, suggesting that Rac1 activity must be regulated tightly for normal axonogenesis. Growth cone morphology was particularly sensitive to dn-RhoA and wt-Cdc42 constructs. These also caused targeting errors, such as tectal bypass, suggesting that cytoskeletal rearrangements are involved in target recognition and are transduced by these pathways.

We report that the growth cones of Xenopus retinal ganglion cells express fibroblast growth factor receptors (FGFRs) and that bFGF stimulates neurite extension from cultured retinal neurons. Furthermore, bFGF is abundant in the developing optic tract but is reduced in the optic tectum. To test whether FGF signaling plays a role in axonal guidance in vivo, bFGF was exogenously applied to the developing optic pathway in "exposed brain" preparations. FGF-treated retinal axons navigate normally through the optic tract, but the majority veer aberrantly at the tectal border and bypass the target. Our results implicate FGF signaling in target recognition and suggest that diminished levels of bFGF in the tectum cause arriving axons to slow their growth.

Axonal tracts in the vertebrate brain seem to respect domains of homeobox gene expression. To test the role of engrailed in tract formation in the midbrain, we inhibited its expression using antisense (AS) oligonucleotides. Phosphorothioate-modified AS oligos caused navigational errors in both the optic projection (OP) and the intertectal commissure (ITC). These oligos, however, also inhibited bFGF binding to the brain. To determine whether these tract phenotypes were due to inhibition of bFGF function or engrailed expression, we used partially phosphorothioate-modified (pp) oligos, which inhibit engrailed expression but do not affect bFGF binding. These ppAS oligos caused the ITC phenotype but had no effect on the OP. Thus, interference with bFGF function correlates with the OP phenotype, while the ITC phenotype is directly related to engrailed expression.

A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three different Xenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10(8) cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the beta-galactosidase gene into the neural tube lumen of 24-h embryos yielded beta-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer in Xenopus embryos and cell lines.

The TCR-CD3 complex consists of the clonotypic disulfide-linked TCRalphabeta or TCRdeltagamma heterodimers, and the invariant CD3delta, epsilon, gamma and zeta chains. We generated plasmid constructs expressing the extracellular domains of the CD3delta, epsilon or gamma subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3delta, epsilon or gamma subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3epsilon-Ig with either the CD3delta-Ig (CD3epsilondelta-Ig) or the CD3gamma-Ig (CD3epsilongamma-Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3epsilondelta-Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3epsilondelta-Ig and anti-CD3 mAb ranged from 10(-8) to 10(-9) M. Based on these results, a single-chain (sc) construct encoding the CD3delta chain linked to the CD3epsilon chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3deltaepsilon-scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3epsilondelta-Ig at a higher affinity than CD3epsilongamma-Ig, suggesting potential structural differences between the CD3epsilondelta and CD3epsilongamma subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR-CD3 complex, and to identify molecules that can interfere with TCR-CD3-mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.