Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain

Abstract

Three major axon pathways cross the midline of the vertebrate forebrain early in embryonic development: the postoptic commissure (POC), the anterior commissure (AC) and the optic nerve. We show that a small population of Gfap+astroglia spans the midline of the zebrafish forebrain in the position of, and prior to, commissural and retinal axon crossing. These glial `bridges' form in regions devoid of the guidance molecules slit2 and slit3,although a subset of these glial cells express slit1a. We show that Hh signaling is required for commissure formation, glial bridge formation, and the restricted expression of the guidance molecules slit1a, slit2, slit3 and sema3d, but that Hh does not appear to play a direct role in commissural and retinal axon guidance. Reducing Slit2 and/or Slit3 function expanded the glial bridges and caused defasciculation of the POC, consistent with a `channeling' role for these repellent molecules. By contrast, reducing Slit1a function led to reduced midline axon crossing, suggesting a distinct role for Slit1a in midline axon guidance. Blocking Slit2 and Slit3, but not Slit1a, function in the Hh pathway mutant yot (gli2DR) dramatically rescued POC axon crossing and glial bridge formation at the midline, indicating that expanded Slit2 and Slit3 repellent function is largely responsible for the lack of midline crossing in these mutants. This analysis shows that Hh signaling helps to pattern the expression of Slit guidance molecules that then help to regulate glial cell position and axon guidance across the midline of the forebrain.

Frizzled-3a and Wnt-8b genetically interact during forebrain commissural formation in embryonic zebrafish

“…In fzd3a loss-of-function embryos the glia and axon defects are rescued by knock down of Slit2, indicating that Fzd3a was indirectly affecting axon guidance. These conclusions are supported by previous reports that Slit2 is essential for glial bridge formation and subsequent axon growth across the midline (Barresi et al, 2005). By performing simultaneous partial knock downs of Wnt8b and Fzd3a we have demonstrated here that these two genes are genetically interacting to regulate commissure formation in the anterior forebrain.…”

“…Slit2 is expressed by a narrow wedge of midline neuroepithelial cells in the commissural plate (Barresi et al, 2005). This expression pattern was not affected by injection of 1ng Std-cont MO (Fig.…”

“…Since GFAP-positive glia and commissural axons are known to be repelled by Slit2 expressed at the midline (Barresi et al, 2005), we next examined the effect of knock down of Wnt8b on slit2 expression by in situ hybridisation. Slit2 is expressed by a narrow wedge of midline neuroepithelial cells in the commissural plate (Barresi et al, 2005).…”

“…Embryos injected with 1ng of wnt8b MO showed an absence of the anterior commissure (unfilled arrowhead, cf. (Barresi et al, 2005).…”

“…The absence of Fzd3a increased the expression of the chemorepulsive ligand slit2 within the commissural plate, which in turn affected the formation of a substrate of glial cells. Glial cells normally form bridges across the midline, facilitating the crossing of commissural axons (Barresi et al, 2005;Barresi et al, 2010). The loss of these bridges leads to aberrant commissure formation.…”