DOI: 10.5061/dryad.wm37pvn0h
title: Fluorescence images of ybx1 mutant neuromasts [Ybx1 immunofluorescence]

description: 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.
description: For the immunolabeling of Ybx1 in wild-type, heterozygous, and
 homozygous <em>ybx1</em> 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 <em>Tg(atoh1a:dTomato)</em> 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.
description: # Fluorescence images of ybx1 mutant neuromasts [Ybx1 immunofluorescence]
 Dataset DOI: [10.5061/dryad.wm37pvn0h](10.5061/dryad.wm37pvn0h) This study
 is comprised of four datasets as follows: * Fluorescence images of ybx1
 mutant neuromasts [incross] (DOI: 10.5061/dryad.v15dv4257): Neuromasts
 from wild-type, heterozygous, and homozygous mutant larvae during
 development and regeneration * Fluorescence images of ybx1 mutant
 neuromasts [time course, wt] (DOI: 10.5061/dryad.g1jwstr1z): Time-course
 images of fixed neuromasts from wild-type larvae * Fluorescence images of
 ybx1 mutant neuromasts [time course, het] (DOI: 10.5061/dryad.jh9w0vtn5):
 Time-course images of fixed neuromasts from heterozygous mutant larvae *
 Fluorescence images of ybx1 mutant neuromasts [Ybx1 immunofluorescence]
 (DOI: 10.5061/dryad.wm37pvn0h): Immunostaining images in neuromasts from
 wild-type and heterozygous mutant larvae ## Description of the data and
 file structure This data set contains fluorescence images of fixed
 neuromasts from outcrossed *ybx1* mutant and *Tg(atoh1a:dTomato)*
 zebrafish larvae. Each image comprises: 1) an `nd` file with parameters,
 *e.g.*, X/Y resolution; and, 2) three-channel fluorescence images, showing
 DAPI (405/460 nm), dTomato (561/600 nm), and anti-Ybx1 immunostaining
 (488/525 nm) signals. ### Files and variables #### File:
 ybx1_het_x_atoh1a_dtom_IF.tar **Description:** Main directory with
 sub-directories for each genotype * Sub-directories are labeled by *ybx1*
 genotype (wild-type [`wt`] and heterozygous mutant [`het`]) * Files are
 labeled with numerical identifiers for unique larvae (`zf`) and neuromast
 (`nm`) * Imaging parameters: &gt; Exposure = 700 ms &gt; WaveName1 =
 "iSIM_561_600/50" &gt; WaveName2 = "iSIM_488_525" &gt;
 WaveName3 = "iSIM \_405_460" &gt; *MagNA* = 1.3 &gt; *MagRI* =
 1.406 &gt; *MagSetting* = 60X SiOil &gt; Width: 154.349 microns (2015)
 &gt; Height: 106.0910 microns (1385) &gt; Depth: 25.5 microns (51) &gt;
 Resolution: 13.0548 pixels per micron &gt; Voxel size: 0.0766x0.0766x0.5
 micron^3