McFarlane Lab
Dr. Sarah McFarlane
Experimental Approaches
One of the most important findings in the past decase is the remarkable degree to which molecular sequences and functions are evolutionarily conserved. Because complex biological pathways are invariably better understood and are more experimentally approachable in simpler organisms, we use Xenopus laevis as our model.
What advantages are offered? Speed, in that the visual system develops in only 3 days. Access, in that embryogenesis occurs ex utero, and the entire process of axon and dendrite outgrowth is available for experimentation. Tight temporal and spatial control, so that genes important early in embryonic development can be the late role of genes in neuronal differentiation can be evaluated. Manipulability, in that we have experimental methods to test the involvement of specific molecules in axon guidance:
1) Gene transfer techniques in vitro and in vivo (McFarlane et al.,1996 ;1998 ;Patel and McFarlane, 2000):
Gene transfer is fundamental to our research examining the role of specific molecules in RGC differentiation. Retinal precursors and RGCs can be made to express foreign genes either in vivo or in vitro using several different techniques.
Transfected Retinal Cells Expressing GFP
2) Pharmacological manipulations in vitro and in vivo ( McFarlane et al., 1995 ;2000 ; Ferguson and McFarlane, 2002; Webber et al., 2002; Webber et al., 2003)
In the growth cone turning culture assay a molecule is released a glass pipette positioned at a distance from an extending growth cone. The concentration will be high at the pipette and low at the growth cone. If the axon reorients towards or away from the pipette it suggests that the molecule acts chemotropically to influence axon behavior. The exposed brain preparation has the advantage of combining an in vivo CNS preparation with the accessibility of a culture dish. The skin and dura overlying the optic tract of embryos are removed, exposing RGC growth cones to applied reagents. Axons are subsequently visualized by anterograde horseradish peroxidase (HRP) labeling.
