Abstract
In contrast to the adult mammalian central nervous system (CNS), fish are able to functionally regenerate severed axons upon injury. Although the zebrafish is a well-established model vertebrate for genetic and developmental studies, its use for anatomical studies of axon regeneration has been hampered by the paucity of appropriate tools to visualize re-growing axons in the adult CNS. On this account, we used transgenic zebrafish that express enhanced green fluorescent protein (GFP) under the control of a GAP-43 promoter. In adult, naïve retinae, GFP was restricted to young retinal ganglion cells (RGCs) and their axons. Within the optic nerve, these fluorescent axons congregated in a distinct strand at the nerve periphery, indicating age-related order. Upon optic nerve crush, GFP expression was markedly induced in RGC somata and intra-retinal axons at 4 to at least 14 days post injury. Moreover, individual axons were visualized in their natural environment of the optic nerve using wholemount tissue clearing and confocal microscopy. With this novel approach, regenerating axons were clearly detectable beyond the injury site as early as 2 days after injury and grew past the optic chiasm by 4 days. Regenerating axons in the entire optic nerve were labeled from 6 to at least 14 days after injury, thereby allowing detailed visualization of the complete regeneration process. Therefore, this new approach could now be used in combination with expression knockdown or pharmacological manipulations to analyze the relevance of specific proteins and signaling cascades for axonal regeneration in vivo. In addition, the RGC-specific GFP expression facilitated accurate evaluation of neurite growth in dissociated retinal cultures. This fast in vitro assay now enables the screening of compound and expression libraries. Overall, the presented methodologies provide exciting possibilities to investigate the molecular mechanisms underlying successful CNS regeneration in zebrafish.
Highlights
The overall projection pattern was comparable to the goldfish, with retinal ganglion cells (RGCs) of one eye sending their axons through the optic nerve and tract to the contralateral optic tectum (Figure 1A)
RGC axons from both eyes crossed completely at the optic chiasm, each optic nerve split into two bundles that interdigitate with the respective bundles of the other side (Figure 1D; Mogi et al, 2009)
The current study characterizes GAP43:green fluorescent protein (GFP) zebrafish with respect to GFP expression in the adult naïve as well as injured visual system as a means to establish this transgenic line as a tool for axon regeneration studies
Summary
Adult teleosts have the remarkable ability to functionally regenerate severed axons in the central nervous system (CNS), for example after lesion of the optic nerve or spinal cord (Stuermer et al, 1992; Bernhardt et al, 1996; Becker and Becker, 2007). This stands in stark contrast to the very limited restorative capability of mammals, including humans, upon CNS injuries (Schwab et al, 1993; Fawcett, 2006; Berry et al, 2008; Fischer and Leibinger, 2012). Upon crush or complete transection of the optic nerve, retinal ganglion cells (RGCs) survive and their severed axons regrow through the optic nerve and tract to topographically re-innervate their respective targets in the brain, leading to functional recovery 2–4 weeks after injury (Stuermer et al, 1992; Bernhardt, 1999; McDowell et al, 2004)
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