The regulation of neuroregeneration in zebrafish (Danio rerio): Global gene expression patterns and the role of estrogen and arylhydrocarbon receptor (AhR) signaling

A lack of understanding of the molecular mechanisms essential for neuroregeneration is a major obstacle to the treatment of neural injury and neurodegenerative diseases. In contrast to adult mammals, teleost fish including zebrafish ( Danio rerio) are able to successfully repair an injury to the central nervous system. Following optic nerve crush (ONX) or sham surgery (SHAM), established markers of neuroregeneration of retinal ganglion cell (RGC) axons (growth associated protein, gap43; alpha tubulin 1, tuba1) were measured by quantitative polymerase chain reaction (qPCR) analysis to verify the occurrence and timing of axonogenesis and synaptogenesis. Global gene expression patterns were documented by microarray analysis in zebrafish eye at intervals during neuroregeneration (6 hours--21 days). Of 15,600 total array transcripts, 4,748 were regulated during regeneration. 93% of 42 sequences tested were successfully validated by qPCR, attesting to the accuracy of the arrays. The number and magnitude of changes and the gene ontology of transcripts were functions of time post-ONX. Regulated sequences represented genes controlling cell adhesion, apoptosis, cell cycle activity, energy metabolism, and calcium signaling. Comparison of our dataset with published transcriptional profiles revealed cell types in eye other than RGCs have roles in neuroregeneration, and indicate an overlap with genes regulated in other regenerating zebrafish tissues. Investigation of regulatory pathways known to be neurotrophic/neuroprotective (estrogen-mediated) or neurotoxic (dioxin-mediated) identified gene markers of exposure and effect. Specifically, expression of aromatase and estrogen receptor (ER) genes was up-regulated very early in regeneration. Treatment with aromatase or ER inhibitors suppressed neuroregeneration-associated genes (gap43) and revealed novel downstream estrogen targets (grn1, sox11, stc2). Potential ER-dependent and -independent effects of estrogen were suggested. Conversely, genes known to mediate dioxin actions (aryl hydrocarbon receptor, AhR) and effects (cyp1a, cyp1b1) were down-regulated during regeneration and dioxin reversed effects of ONX on regeneration-associated markers ( tuba1, wnt2, spond1, sox11). Results reported in these studies suggest a physiological role for AhR during neuroregeneration and potential crosstalk between AhR and estrogen signaling pathways. Results of this research advance the zebrafish model for studying molecular processes of neuroregeneration and provide an entry point for better understanding the underlying regulatory and toxic mechanisms.