Fig 3. Small molecule screen workflow.

For simplicity a single 96-well plate containing active compounds is shown. First we tested the toxicity of the individual compounds on larval zebrafish and excluded 134 toxic compounds from further analysis (black wells). We chose to apply the compounds at a uniform dilution of 1:1,000 since this ensured a fast throughput and a relatively low percentage of toxic compounds. Depending on the stock concentration, most compounds were tested at a final concentration of 1 μM or 5 μg/ml with some exceptions (S1 Table). 346 remaining compounds were assigned to 69 pools. Here, examples of two pools are shown (yellow and orange). In a first pass, we tested the 69 compound pools for their effect on nerve regrowth in the fin removal assay and found 15 pools that affected nerve regrowth (differently colored pools). Then we tested the individual compounds from these 15 pools in a second pass and identified 21 individual compounds that affected nerve regrowth in the fin removal assay. Ten of these compounds were further tested using laser nerve transection. Timeline diagrams of treatment after fin removal and laser nerve transection are shown on the right side.