Autonomous and non-autonomous Shh signalling mediate the in vivo growth and guidance of mouse retinal ganglion cell axons

Abstract

In non-mammalian vertebrates, the relatively homogeneous population of retinal ganglion cells (RGCs) differentiates and projects entirely to the contralateral side of the brain under the influence of sonic hedgehog (Shh). In mammals, by contrast, there are two different RGC types: the Zic2-positive ipsilateral projecting and the Isl2-positive contralateral projecting. We asked whether the axons of these two populations respond to Shh and if their response differs. We have also analysed whether midline-and RGCderived Shh contributes to the growth of the axons in the proximal visual pathway. We show that these two RGC types are characterised by a differential expression of Shh signalling components and that they respond differently to Shh when challenged in vitro. In vivo blockade of Shh activity, however, alters the path and distribution mostly of the contralateral projecting RGC axons at the chiasm, indicating that midline-derived Shh participates in funnelling contralateral visual fibres in this region. Furthermore, interference with Shh signalling in the RGCs themselves causes abnormal growth and navigation of contralateral projecting axons in the proximal portion of the pathway, highlighting a novel cell-autonomous mechanism by which Shh can influence growth cone behaviour.

Segregation of Ipsilateral Retinal Ganglion Cell Axons at the Optic Chiasm Requires the Shh Receptor Boc

“…However, as Shh activity is important to pattern the midbrain regions surrounding the ventral hypothalamus (just adjacent to the optic chiasm) during retinal axon development (Dale et al, 1997;Barresi et al, 2005;Manning et al, 2006), it is likely that blocking Shh signaling for 5 d in developing embryos can cause patterning defects. Thus, as noted by the authors (Sánchez-Camacho and Bovolenta, 2008), it is possible that the effects observed are not due to a direct guidance effect of Shh but rather to changes in expression of other guidance cues. In a second set of experiments, an inhibitor of the Shh signaling mediator Smo was electroporated in the developing mouse visual system at E13.5 and analyzed at E16.5 (Sánchez-Camacho and Bovolenta, 2008).…”

“…Retinal explants isolated from Boc ϩ/Ϫ and Boc Ϫ/Ϫ E15.5 embryos were cultured for 24 h. RGC axon dynamics were analyzed by time-lapse microscopy after addition of 10 nM ShhN. (Trousse et al, 2001;Kolpak et al, 2005;Sánchez-Camacho and Bovolenta, 2008) and it is present in the chiasmatic zone (Torres et al, 1996;Marcus et al, 1999;Wallace and Raff, 1999;Trousse et al, 2001). Based on these data and on our finding that Boc is expressed in ipsilateral RGCs, we hypothesized that Boc might function as a Shh receptor which prevents these axons from crossing the optic chiasm.…”

“…We thus investigated whether Shh could control the behavior of ipsilateral RGC axons. Many of the experiments implicating a role for Shh in the guidance of RGC axons were performed over a long time period (24 -48 h) and therefore did not distinguish between short-term, direct effects and long-term, indirect effects of Shh on the growth cone of these axons (Trousse et al, 2001;Kolpak et al, 2005;Sánchez-Camacho and Bovolenta, 2008). Moreover, it is not clear whether Shh has a direct effect on all RGC axons, or only on a subpopulation of RGCs.…”

“…In addition to in vitro experiments, in vivo experiments have also suggested a role for Shh in mouse RGC axon pathfinding (Sánchez-Camacho and Bovolenta, 2008). Injection of E13.5 mouse embryos with an hybridoma producing a Shh-blocking antibody (and analyzed at E18.5) alters the distribution of RGC axons at the optic chiasm with an enlargement of the overall region occupied by RGC fibers at the midline and axon extension in aberrant directions.…”

“…Shh is expressed in the diencephalic ventral midline (Torres et al, 1996;Marcus et al, 1999;Wallace and Raff, 1999;Trousse et al, 2001) and it has been suggested to act as a repellent to guide RGC axons through the optic chiasm (Trousse et al, 2001). More recently, in vivo experiments that inhibit the Shh signaling mediator Smoothened (Smo) in the developing mouse visual system suggested that Shh controls the pathfinding of contralaterally projecting RGC axons in a cellautonomous way (Sánchez-Camacho and Bovolenta, 2008). However, contralaterally and ipsilaterally projecting RGCs both express components of the Shh signaling pathway, and these experiments did not assess whether ipsilaterally projecting RGC axons are normally guided at the chiasm under these conditions.…”