Fluorescence images of ybx1 mutant neuromasts [Ybx1 immunofluorescence]

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
For the immunolabeling of Ybx1 in wild-type, heterozygous, and homozygous ybx1 mutant larvae, we fixed 5 dpf zebrafish trunks in 4% formaldehyde for 3-4 hr at room temperature, then rinsed with methanol prior to storage at -20 °C. Before immunostaining, we gradually rehydrated the trunks in fresh PBS, permeabilized them for 5 m with cold acetone (-20 °C), then washed them several times in 0.1 % PBST. We incubated the trunks at room temperature for 2-4 hr or overnight at 4 °C in a 0.1% PBST blocking solution containing 5 % normal donkey serum and 2 % DMSO, then again overnight at 4 °C in fresh blocking solution with rabbit anti-YB-1 (C-terminal) antibodies (1:100; Sigma-Aldrich, St. Louis, USA). After several washes in 0.1 % PBST, we again incubated the trunks for 3 hr at room temperature in fresh blocking solution with donkey Alexa Fluor 488 anti-rabbit antibodies (1:500; Invitrogen, Eugene, USA). After further washes in 0.1 % PBST, we incubated the trunks for 10 m in DAPI (1:500) and stored them at 4 °C until imaging them at 60X magnification. When immunostaining Tg(atoh1a:dTomato) larvae, we additionally used monoclonal mouse anti-RFP antibodies (1:1000; Invitrogen, Eugene, USA) and donkey Alexa Fluor 561 anti-mouse antibodies (1:500; Invitrogen, Eugene, USA) during the primary and secondary incubations, respectively.