Axon Guidance in the Forebrain
A large scale screen for retinotectal mutants identified a large number of mutants that affect the ability of axons to cross the midline of the forebrain, and no optic chiams forms (Karlstrom et al, 1996).  Many of these  mutations affect the Hedgehog/Gli signaling pathway and provide tools for understanding how Hh/Gli signaling establishes the axon growth substrate (Barresi et al, 2005).  We recently showed that the belladonna (bel) mutation affects the lhx2 gene (Seth et al, 2006).  bel(lhx2) appears to specifically affect guidance of only a few axons in the forebrain.  We found that glial cells "bridge' the midline just prior to axon crossing, and this glial bridge is affected in the Hh pathway mutants as well as bel(lhx2).  We are currently investigating the role these glial cells, as well as the guidance molecules of the slit/robo and sema families, affect axon guidance across the midline and help establish the optic chiasm and forebrain commissures.

axon defects in bel(lhx2)
Axon guidance errors in yot(gli2) and bel(lhx2) mutants.  Ventral (facial) views of wildtype (left) and bmutant zebrafish embryos labeled with an antibody to visualize axons. Left: Wildype axons cross the midline of the forebrain to form the optic chiasm (top image), postoptic commissure and anterior commissure. (lower image)  These axons fail to cross the midline in yot (top right)  and bel (bottom right)  mutants.
A 3D look at the Zebrafish Chiasm Region KarlstromLabHomePage  
In the lab we use different imaging techniques in combination with genetics and gene-knock-down approaches to investigate the formation of nerve pathways during embryonic development.  We are focusing on understanding the cellular and molecular cues that help guide axons across the midline of the zebrafish forebrain.  The first axons to cross the midline in this region are commissural axons that form the postoptic commmisure.  Slightly later in development, axons from the eye grow across the same region to form the optic nerve and optic chiasm.
 
fb axon diagram Model of axons and glia in the zebrafish forebrain. 
Red cells are a glial "bridge" that spans the midline
prior to axon crossing.  Purple square indicates the
region shown in the two movies below.  The movies on the right show frontal views of the the entire zebrafish head, eyes on either side.
 30 hours: RGC axons near midline: Confocal movie of axons (green) and  GFAP positive glial cells (red) at 30 hrs.  Retinal axons have just grown into the brain.  Click to see 3D movie of this image.  Movie by M. Barresi
3D rotating image of glial cells (red), POC
commissural axons (bottom green band) and retnial axons
(top greeen band) in the diencephalon.  Click on the image
to see a rapidly rotating 3D movie of this data set. 
Deconvolution software (Openlab) was used to improve the
image, allowing us to view details of glial cell morphology
not otherwise visible. Movie by M. Barresi.

 48 hours: A well formed chiasm. Confocal movie of axons (green) and  GFAP positive glial cells (red) at 48 hrs.  At this age, retinal axons have crossed the midlien to form the optic chiasm.  Click to see 3D movie of this image.Movie by M. Barresi
Confocal movie showing commissural axons (red) growing in
relation to cells expressing a Sema3D-GFP fusion protein
(green) under the control of an HSP70, heat-shock inducible, promoter. Embryos were injected with an HSP70-Sema3D-GFP plasmid construct at the 2 cell stage to produce an embryo with mosaic inheritence of the plasmid.  Embryos were grown to 19 hours, heat-shocked for 50 minutes to induce Sema3D-GFP
expression, then grown to 24 hours when they were fixed
and labeled with  an antibody to show growing axons (red). 
These images were acquired on a Zeiss meta confocal microscope by Anandita Seth in the Karlstrom Lab.  Movie by A. Seth		 48 hours: A well formed chiasm. Confocal movie ofZn-5 labeled retinal ganglion cells and axons (green) at 48 hrs.  Retinal axons have just grown into the brain.  Zn-5 also labels the adenohypophysis (pituitary placode). Click to see 3D movie of this image. Movie by M. Barresi