Fluorescence images of ybx1 mutant neuromasts [time course, wt]

Like the sensory organs of the human inner ear, the lateral-line neuromasts (NMs) of fish such as the zebrafish (Danio rerio) contain mechanosensory hair cells (HCs) that are surrounded by progenitors called supporting cells. Damaged NMs can quickly regenerate new HCs by expressing in the progenitors HC-specific genes such as atoh1a, the master regulator of HC fate. We used the supervised learning algorithm DELAY to infer regenerating NMs’ early gene-regulatory network (GRN) and identify adaptations that promote the rapid regeneration of lateral-line HCs in larval zebrafish. The central hub in the network, Y-box binding protein 1 (ybx1), is highly expressed in HC progenitors and young HCs and can recognize DNA-binding motifs in cyprinids’ candidate regeneration-responsive promoter elements for atoh1a. We showed that NMs from ybx1 mutant zebrafish larvae display consistent, regeneration-specific deficits in HC number and initiate both HC regeneration and atoh1a expression 20 % slower than in siblings. By demonstrating that ybx1 promotes rapid HC regeneration through early atoh1a upregulation, the results support DELAY’s ability to identify key temporal regulators of gene expression.

Methods
To label HCs, we fixed 3-5 dpf zebrafish trunks with 4% formaldehyde in phosphate-buffered saline (PBS) solution with 0.1 % Tween-20 (0.1 % PBST) for either 1 hr at room temperature or overnight at 4 °C. For Tg(atoh1a:dTomato) larvae, we washed trunks in 0.5 % PBST and incubated them for 1 hr at room temperature in 0.05 % PBST blocking solution with 1 % BSA and again for 2 hr in fresh blocking solution with DAPI (1:200), phalloidin (1:40) and goat anti-tdTomato DyLight550-conjugated antibodies (1:50; MyBioSource, San Diego, USA). We washed trunks once more for 20-30 m in 1X PBS before mounting and imaging at 100X on an Olympus IX83 inverted confocal microscope with a microlens-based super-resolution imaging system (VT-iSIM, VisiTech International).