Identification of Modulators of Hair Cell Regeneration in the Zebrafish Lateral Line

Abstract

The external location of the zebrafish lateral line makes it a powerful model for studying mechanosensory hair cell regeneration. We have developed a chemical screen to identify FDA-approved drugs and biologically active compounds that modulate hair cell regeneration in zebrafish. Of the 1,680 compounds evaluated, we identified 2 enhancers and 6 inhibitors of regeneration. The two enhancers, dexamethasone and prednisolone, are synthetic glucocorticoids that potentiated hair cell numbers during regeneration and also induced hair cell addition in the absence of damage. BrdU analysis confirmed that the extra hair cells arose from mitotic activity. We found that dexamethasone and prednisolone, like other glucocorticoids, suppress zebrafish caudal fin regeneration, indicating that hair cell regeneration occurs by a distinctly different process. Further analyses of the regeneration inhibitors revealed that two of the six, flubendazole and topotecan, significantly suppress hair cell regeneration by preventing proliferation of hair cell precursors. Flubendazole halted support cell division in M-phase, possibly by interfering with normal microtubule activity. Topotecan, a topoisomerase inhibitor, killed both hair cells and proliferating hair cell precursors. A third inhibitor, fulvestrant, moderately delays hair cell regeneration by reducing support cell proliferation. Our observation that hair cells do not regenerate when support cell proliferation is impeded confirms previous observations that cell division is the primary route for hair cell regeneration after neomycin treatment in zebrafish.

Auditory sensitivity of larval zebrafish (Danio rerio) measured using a behavioral prepulse inhibition assay

“…Larvae at 5d.p.f. were exposed to 400μmoll −1 neomycin in embryo medium for 1h and then immediately rinsed four times in fresh embryo medium (Harris et al, 2003;Murakami et al, 2003;Owens et al, 2009;Namdaran et al, 2012). Neomycin exposure at concentrations greater than 100μmoll −1 is known to significantly reduce swimming speed in larval zebrafish (Buck et al, 2012).…”

An Assay for Lateral Line Regeneration in Adult Zebrafish

“…Based on the extensive body of literature that has been established for analysis of lateral line (LL) regeneration in embryonic and larval zebrafish 8,24,25 In regard to the number of neuromasts along the lateral line of zebrafish, embryonic and larval zebrafish have the distinct advantage that the neuromasts have a simple pattern because of their lower number within a given stitch as compared to adult fish. This low number has allowed investigators to clearly identify each neuromast and assign a name to it 6 .…”

“…A limitation of the assay concerns the affect of the experimental condition whether it be 1) the transgenic strain that mimics a particular disease state or 2) a pharmacologically-induced disease state] on stem cells within the lateral line system of adult zebrafish. In this regard, a particular disease state of the adult zebrafish may or may not affect stem cells of the hair cell lineage, and it is important to note that neuromast regeneration is completely dependent on these stem cell proliferation/differentiation processes 8,24,25 .…”

Phenotypic Screens Targeting Neurodegenerative Diseases

“…Several successful examples have been provided in the previous sections, such as the C. elegans or Zebrafish screens. 45,49,62 One powerful screening approach is to use genetic manipulation, such as GFP labeling, to score the desired phenotype. However, there are several complications with these screening models, such as the species differences of target proteins, the difficulty of achieving assay robustness due to inconsistent compound exposure and culture preparations, and the limited assay throughput and analytical methodologies.…”