Abstract
Purpose :
Unlike mammals, zebrafish successfully regenerate their optic nerve (ON) and recover vision after injury. The exact signaling mechanisms mediating their efficient axon regeneration are unknown. We used spatial transcriptomics to reveal the differential expression of genes across the healthy and regenerating retinal ganglion cells (RGCs), ONs, tracts (OT), and optic tecta (OTe). We have begun to test the role these identified genes and pathways play in ON regeneration.
Methods :
Tissue was harvested 3 days after optic nerve crush from adult zebrafish. Eyes and ON, OT, and OTe were carefully dissected. Eyes were processed for laser capture microdissection sequencing of the RGCs while intact ON, OT, and OTe tissue was prepared for spatial transcriptomics using the 10x Visium system. Analysis was performed using 10x Genomics Space Ranger and Loupe Browser as well as standard bulk-seq methods. RNAscope in-situ hybridization (ISH) was used to validate differential gene expression. To test the functional relevance of identified genes we chose to inhibit the function of the kitb and pdgfra receptors using antisense morpholinos and chemical inhibitors. Axon regeneration into the OTe was measured in control and treated groups using the gap43:GFP reporter line at 7 days post injury.
Results :
K-means clustering revealed 10 regions of differential gene expression across the ON, OT, and OTe, suggesting that the injury site has a distinct expression profile from the rest of the injured and control ON. The OT and OTe of the injured side are also distinct from the uninjured control side. kitb and pdgfra were identified as highly induced in RGCs after injury and their ligands, kitlgb and pdgfaa, upregulated in ON, OT, and OTe. Chemical or genetic inhibition of kitb and pdgfra resulted in significantly decreased regeneration which was enhanced by co-knock down suggesting redundancy in function. ISH for kitlgb and pdgfaa, surprisingly shows that they are upregulated in distinct cell types following injury.
Conclusions :
The response to ON injury results in distinct regional changes in gene expression within the visual system during ON regeneration in zebrafish. Spatial transcriptomic data from this model can be used to identify ligand-receptor pairs, Kitlgb-Kitb and Pdgfra-Pdgfaa, that mediate successful ON regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.