Drug screen in a zebrafish model of NEB-related nemaline myopathy. Schematic depicting the experimental flow of an unbiased drug screen in neb mutant zebrafish using the Microsource Discovery library. Heterozygous neb zebrafish (neb+/−) were mated, and neb mutant (neb−/−) offspring identified by phenotype. Once identified at 2 dpf, single neb−/− embryos were placed into individual wells of a 96-well plate, each containing a single drug at a dose of 10 μm. Embryos were incubated for 16 h, and then analyzed for movement using the Viewpoint Zebrabox. All drugs were tested in duplicate. Positive hits, as defined using a Z analysis of the movement data, were re-tested using n=16 neb (−/−) zebrafish and multiple doses of the drug. While eight drugs were initially identified as positive hits, no single compound gave a positive result upon retesting.

neb^−/− embryos exhibit decreased mobility and survival relative to their WT siblings. At 3 dpf, neb^−/− embryos (n=25) displayed impaired movement both in terms of total time spent moving (A) and total distance travelled (B) relative to their wild-type (WT) siblings (n=25) when tested with the Viewpoint Zebrabox. (C) neb^−/− embryos also exhibit dramatically lower viability relative to WT siblings, with most dying by 6 dpf. ****P<0.0001.

ENU-based suppressor screen in a mousemodel of NEB-related NM. Schematic of dominant suppressor screen. Heterozygous male Neb mutant (Neb^Δ55/+) mice were treated with ENU (90 mg/kg weekly for 3 weeks), then allowed to recover for up to 8 weeks, then tested for fertility by mating with female WT CD-1 animals. Fertile males were then mated on a rotating schedule with heterozygous Neb mutant (Neb^Δ55/+) females. The resulting generation one (G1) offspring were monitored daily starting from birth. A subset of live-born pups exhibited an obvious phenotype consistent with the reported Neb phenotype. These animals all died by P7. Animals surviving beyond this point were monitored daily, and then genotyped by age 3 weeks. No animals surviving to 3 weeks had the Neb mutant genotype (Neb^Δ55/Δ55). 14 G1 heterozygous mice unexpectedly had weakness (heterozygous mice are typically normal). However, upon inheritance testing the motor dysfunction phenotypes did not segregate with the heterozygous genotype. Of note, there was non-significant (P=0.0582) skewing of numbers of wild-type versus heterozygous mice, potentially indicating that some heterozygous mice had a second Neb mutation in trans that resulted in a perinatal lethal phenotype similar to that of Neb^Δ55 homozygotes.