© 2015. Published by The Company of Biologists Ltd | Biology Open (2015) 4, 903-909 doi:10.1242/bio.

012112

RESEARCH ARTICLE

Inexhaustible hair-cell regeneration in young and aged zebrafish

Filipe Pinto-Teixeira‡,§, Oriol Viader-Llargué s*,§, Elen Torres-Mejı́a*, Melissa Turan, Estela Gonzá lez-Gualda,
Laura Pola-Morell* and Herná n Ló pez-Schier*,¶

ABSTRACT Some 16–20 mechanosensory hair cells occupy the center of the
Animals have evolved two general strategies to counter injury organ, whereas the remainder are two types of non-sensory
and maintain physiological function. The most prevalent is protection supporting cells: around 30 sustentacular cells intermingle with
by isolating vital organs into body cavities. However, protection is not hair cell, and fewer than 10 mantle cells outline the organ
optimal for sensory systems because their external components (Fig. 1A). Interneuromast cells connect each organ. Lateralis hair
need to be exposed to the environment to fulfill their receptive function. cells are born in pairs from the terminal mitotic division of
Thus, a common strategy to maintain sensory abilities against unipotent hair-cell progenitors (UHCPs). The neuromast has a
persistent environmental insult involves repair and regeneration. stereotypical symmetry formed by one axis of planar cell polarity
However, whether age or frequent injuries affect the regenerative and by the position of functionally distinct non-sensory equatorial
capacity of sensory organs remains unknown. We have found that and polar areas (Fig. 1A). Equatorial cells are under sustained
neuromasts of the zebrafish lateral line regenerate mechanosensory Notch signaling, which restricts the development of UHCPs to
hair cells after recurrent severe injuries and in adulthood. Moreover, permissive polar areas with low Notch (Wibowo et al., 2011).
neuromasts can reverse transient imbalances of Notch signaling that Stem cells have not been described in neuromasts, and whether the
result in defective organ proportions during repair. Our results reveal regenerative capacity of neuromasts diminishes with age or after
inextinguishable hair-cell regeneration in the lateral line, and suggest recurrent damage remains unknown.
that the neuromast epithelium is formed by plastic territories that are
maintained by continuous intercellular communication. RESULTS AND DISCUSSION
The ET(krt4:EGFP)sqgw57A transgenic line highlights
KEY WORDS: Adult, Hair cells, Lateral line, Notch, Regeneration, Sox-2+ cells in neuromasts
Self organization To assay neuromast architecture we acquired a collection of
fluorescent transgenic lines with complementary expression
INTRODUCTION patterns. As shown previously, the green-fluorescent line
Sensory receptors are the interphase between the environment and Tg[Cldnb:mem-EGFP] highlights the whole neuromast and the
the nervous system. Vertebrates are generally able to repair sensory interneuromast cells, and weakly the peridermal cells (Fig. 1B)
organs, but mammals cannot regenerate the hair cells of their inner (Haas and Gilmour, 2006; López-Schier and Hudspeth, 2006).
ear (Burns and Corwin, 2013; Groves et al., 2013; Rubel et al., The Tg[ET(krt4:EGFP)sqet20] line marks interneuromast cells
2013). Consequently, ototoxic antibiotics, anti-neoplastic and highlights the equatorial areas (Fig. 1C, supplementary material
therapies or high levels of sound cause irreversible hearing loss Fig. S1) (López-Schier and Hudspeth, 2006; Parinov et al.,
and balance disorders (Chen and Fechter, 2003; Huth et al., 2011; 2004), whereas the red-fluorescent Tg[Alpl:mCherry] is expressed
Langer et al., 2013). Hair cells in fishes and amphibians occurring homogeneously in the peripheral cells of the neuromast and
in the ear and the lateral line are also susceptible to damage by in interneuromast cells (Fig. 1C, supplementary material Fig. S1)
overstimulation or by drug-induced ototoxicity (Harris et al., (Steiner et al., 2014). Tg[ET(krt4:EGFP)sqet4] expresses EGFP
2003; Jiang et al., 2014; López-Schier and Hudspeth, 2006; Ma in the UHCPs and hair cells (Fig. 1B) (López-Schier and
et al., 2008; Millimaki et al., 2010; Schuck and Smith, 2009; Hudspeth, 2006; Parinov et al., 2004; Wibowo et al., 2011),
Steiner et al., 2014). However, these aquatic vertebrates can and the Tg[ pou4f3:gap43-GFP] only marks the hair cells (Fig. 1D)
rapidly regenerate hair cells (Behra et al., 2009; Hernández et al., (Xiao et al., 2005). Next, we established a new transgenic line
2007; Williams and Holder, 2000). The superficial lateral line of called Tg[ET(krt4:EGFP)sqgw57A] to better characterize hair-
zebrafish is composed by a collection of neuromasts that are cell regeneration in vivo. It was generated by the genomic insertion
formed by a simple circular epithelium of approximately 60 cells. of a gene-trapping vector carrying a green-fluorescent protein
(Kondrychyn et al., 2011). We found that Tg[ET(krt4:EGFP)
Laboratory of Sensory Cell Biology & Organogenesis, Centre for Genomic sqgw57A] expresses EGFP in Sox-2+ cells, but not in interneuromast
Regulation, Dr. Aiguader 88, 08003 Barcelona, Spain.
*Present address: Helmholtz Zentrum Mü nchen, German Research Center for
cells or hair cells (Fig. 1E-G). Sox-2 is a transcription factor at the
Environmental Health, Ingolstaedter Landstraße 1, D-85764 Neuherberg, Munich, apex of the gene-expression cascade that establishes sensory
Germany. competence in the neuroepithelium at the earliest stages of hair-
‡
Present address: New York University, 70 Washington Square South, New York,
NY 10012, USA. cell development (Kiernan et al., 2005; Millimaki et al., 2010;
Biology Open

§
These authors contributed equally to this work Neves et al., 2013). In the zebrafish lateral line and inner ear, cells
¶ expressing Sox-2 are the source of hair-cell progenitors (Hernández
Author for correspondence (hernan.lopez-schier@helmholtz-muenchen.de)
et al., 2007; Millimaki et al., 2010). Therefore, Tg[ET(krt4:EGFP)
This is an Open Access article distributed under the terms of the Creative Commons Attribution sqgw57A] is likely to highlight the cells that will be canalized to a
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed. UHCPs fate in permissive polar areas. This comprehensive
collection of transgenic lines allows the unambiguous visualization
Received 20 March 2015; Accepted 11 May 2015 of cell identity, distribution, and number in neuromasts (Fig. 1H).

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adult (1- and 2-year old) stages with neomycin. In all cases, hair-cell
regeneration occurred within 72 hpt (Fig. 2A-C, and data not
shown). Using 1-year old adult fish in which the Tg[myo6b:actb1-
EGFP] transgene reveals the apical hair bundle of the hair cells
(Fig. 2D-F), and 6-month old Tg[Alpl:mCherry ; ET(krt4:EGFP)
sqet20] that shows neuromast geometry (Fig. 2G-H), we found that
cell polarity and epithelial architecture were comparable between
controls and neomycin-treated samples 72 hpt. Thus, neuromasts
are endowed with invariant and enduring regenerative capacity,
which may have evolved for fish to maintain life-long sensory
ability despite persistent environmental insult (Ciba-Foundation,
1991).

Hair-cell regeneration is unaffected by the recurrent and

frequent loss of hair cells
After treatment with neomycin, the first hair cells appear around
8 hpt, with a sequential production of pairs of hair cells up to 20 by
72 hpt (Ma et al., 2008; Wibowo et al., 2011; Lush and Piotrowski,
2014). We hypothesized that the fast onset of regeneration can be
explained by the presence of a subpopulation of “primed” cells that
are quickly routed towards a UHCP fate. Therefore, a frequent and
long sequence of hair-cell ablations should deplete the epithelium
from primed cells, leading to regenerative decline. To test this idea
we subjected 2-year old transgenics Tg[ET(krt4:EGFP)sqet4] to six
consecutive neomycin treatments with intervening 24-h periods of
rest between treatments to allow partial regeneration. Hair cells
regenerated efficiently after the sixth injury cycle (Fig. 3A-B). To
assess the temporal profile of regeneration, we subjected Tg[ET
(krt4:EGFP)sqet4] larvae to six consecutive hair-cell ablations with
neomycin and counted hair cells 24 h after each treatment.
Neuromasts showed invariable hair-cell regeneration after each
ablation (Fig. 3C-D). Thus, to maintain this inexhaustible
regenerative capacity at constant kinetics, each organ must
persistently produce at least 4 UHCPs per day, representing over
10% of the originating Sox-2+ cells.

Atoh1a is transiently and broadly expressed in the

epithelium
Hair cells require Atoh1a for their development. Ma and colleagues
showed that Atoh1a is expressed in broad areas of the epithelium
during the first 24 h of regeneration (Ma et al., 2008). Using the
Fig. 1 . Neuromast organization and transgenic zebrafish lines.
double-transgenic line Tg[Atoh1a:dTomato ; ET(krt4:EGFP)sqet4]
(A) Scheme of a neuromast depicting different cell types and axes of
symmetry. (B-F) Confocal image of a larval neuromast of (B) Tg[Cldnb:mem- to simultaneously visualize Atoh1a expression and hair cells, we
EGFP;ET(krt4:EGFP)sqet4] (green), incubated in the vital dye DiAsp to reveal extended these previous observations by finding a broad but weak
functional hair cells (red), (C) Tg[Alpl:mCherry (red) ; ET(krt4:EGFP)sqet20 Atoh1a in supporting cells, which stabilizes and becomes stronger
(green)] in which white arrowhead point to interneuromast cells, (D) Tg[ pou4f3: in UHCPs and in young hair cells (Fig. 3E). This pattern of Atoh1a
gap43-GFP (green) ; pou4f3:Ribeye-Kusabira (red)], (E) Tg[ET(krt4:EGFP) expression substantiates the prediction that neuromasts may use a
sqgw57A] (green) labeled with DiAsp (red), (F) Tg[ET(krt4:EGFP)sqgw57A
large pool of supporting cells to generate UHCPs to sustain
(green) ; Alpl:mCherry (red)], (G) Tg[ET(krt4:EGFP)sqgw57A] (green)
immunostained for Sox-2 (red) and incubated with the nuclear dye DAPI (blue).
regeneration upon recurrent severe damage, and suggests that the
(H) Overview of a neuromast with the different cell types and transgenic expression of Atoh1a occurs by default in Sox-2+ cells. To further
markers. Scale bars=10 µm. test this hypothesis we abrogated Notch signaling in the entire
regenerating neuromast by treating zebrafish larvae with the
Hair cells regenerate efficiently in larval, juvenile and adult γ-secretase inhibitor DAPT (Ma et al., 2008; Wibowo et al.,
zebrafish 2011). The uniform loss of Notch signaling stabilized Atoh1a
A single treatment with the ototoxic aminoglycoside antibiotic in a broader area of the neuromast (Fig. 3F), and generated
Biology Open

neomycin readily ablates every functional hair cell in the superficial supernumerary hair cells ectopically (Fig. 3F-G, supplementary
lateral line of the zebrafish larva (Harris et al., 2003; López-Schier material Fig. S2A-B). The spatial profile of Sox-2 expression and of
and Hudspeth, 2006; Pinto-Teixeira et al., 2013). Subsequently, Notch activity in neuromasts suggests that they do not regulate each
neuromasts enter a regenerative process that is largely complete other (Hernández et al., 2007; Ma et al., 2008; Wibowo et al., 2011).
72 hours post (neomycin) treatment (hpt) (Ma et al., 2008; Wibowo Supporting this conclusion, Sox-2 expression was not qualitatively
et al., 2011). To assess hair-cell regeneration in older animals, we affected upon a constitutive activation of Notch signaling by
treated three different transgenic lines at juvenile (3-month old) and expressing the intracellular domain of Notch (NICD) by neuromast-

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Fig. 2. Efficient hair-cell regeneration in adult zebrafish.

(A-C) Maximal projection of confocal images from Tg[ET(krt4:
EGFP)sqet4] transgenics (green) showing neuromasts of the
caudal fin of a 2-year old fish stained with DAPI (red) (A) before
neomycin-treatment, (B) 2 h after treatment, and (C) 72 h after
treatment. (D-E) Neuromast of an adult fish from Tg[myo6b:
actb1-EGFP] transgenics (green) stained with DAPI (red)
showing (D) hair cells controls, and (E) in neomycin-treated fish
after hair-cell regeneration. (F) XZ profile of the same
neuromast in E showing the apicobasal polarization of the
regenerated hair cells (green). (G-H) Neuromast of an adult fish
from Tg[Alpl:mCherry (red) ; ET(krt4:EGFP)sqet20 (green)]
transgenics stained with DAPI (blue), showing (G) epithelial
geometry in control fish and (H) in neomycin-treated fish at
72 hpt. In all cases, N=8 neuromasts from 2 animals. Scale
bars=10 µm.

specific chemical induction with tamoxifen using the double activity, and may explain the inexhaustible production of hair cells
transgenic line Tg[Cldnb:Gal4ERT2;5xUAS-E1b:6xMYC-notch1a] upon recurrent severe damages.
(Fig. 4A-G). However, we observed a modestly significant
increase of the number of Sox-2+ cells that was accompanied by a Neuromast organization is reversibly affected by imbalances
small but non-significant increase of the total number of cells of Notch signaling
(Fig. 4G). It has been proposed that a feedback inhibition of supporting-cell
During otic development, Sox-2 establishes pro-sensory proliferation via Notch signaling maintains a constant cellular
competence in the neuroepithelium for the generation of neurons population in neuromasts (Ma et al., 2008). Thus, to test a functional
and hair cells by: (1) the direct activation of Atoh1 expression and, link between intercellular communication and organ proportions,
(2) the activation of negative regulators of Atoh1 function (Neves we again examined neuromasts with defective Notch activity.
et al., 2013). In neuromasts, however, two parallel direct inputs We first expressed NICD constitutively by heat-shock in Tg[hsp70l:
appear to control the spatiotemporal mode of Atoh1a expression: a Gal4; 5xUAS-E1b:6xMYC-notch1a], and by chemical induction
constitutive and un-patterned activation by Sox-2, and a patterned with tamoxifen in the transgenic line Tg[Cldnb:Gal4ERT2;5xUAS-
inhibition by Notch. In contrast to the ear, the neuromast E1b:6xMYC-notch1a], which halted hair-cell regeneration almost
epithelium does not produce neurons, which may account for the immediately (Fig. 4H, supplementary material Fig. S2C), indicating
differences between the two organs. The profile of Atoh1a that neuromasts do not contain “engaged” UHCPs that are refractory
expression in normal circumstances and in conditions of high and to Notch inhibition. In a converse experiment, we abrogated Notch
low Notch signaling provides a mechanistic explanation for the signaling by treating zebrafish with DAPT, which increased hair-
sequence and spatiotemporal pattern of sustentacular-cell cell production (Figs 3G, 4I, supplementary material Fig. S2A-B).
conversion into UHCP: the mitotic division of Sox-2+ cells is Total cell counts varied only marginally in DAPT-treated neuromasts
symmetric with regard to the UHCP potential of the two daughter and matched the excess of hair cells (on average, circa 16% of the
cells. However, the independent movement of the daughter cells cells in control neuromasts were hair cells, versus 27% in DAPT-
within the epithelium places them in different signaling treated samples), revealing an imbalance of cell types and epithelial
environments: the cell remaining in an equatorial zone will proportions (Fig. 4G,I-L). Control and DAPT-treated fish were then
continue to be exposed to high levels of Notch signaling and subject to a final incubation in neomycin to ablate hair cells, and
maintain a sustentacular-cell character, whereas those entering a subsequently returned to normal conditions. The neuromasts that had
Biology Open

polar zone with low Notch activity will stabilize Atoh1a been exposed to DAPT continued to produce supernumerary hair
expression and become UHCP. Although we do not know if the cells one day after the second neomycin treatment (Fig. 4L). This
movement of each daughter cell is stochastic or deterministic, a result suggests that after lifting Notch inhibition, a larger than normal
default broad expression of Atoh1a provides a robust mechanism pool of sustentacular cells produces UHCPs, possibly by a lasting re-
for UHCP production regardless of how cells move in the tissue. organization of the spatial influence of Notch. Remarkably, however,
These results reinforce the conclusion that potentially all Sox-2+ these neuromasts recovered almost normal proportions one week later
cells may quickly generate UHCPs upon reduction of Notch (Fig. 3G). Taken together, these results strongly suggest that the

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Fig. 3. Hair-cell regeneration after

recurrent damage. (A-B) Confocal images
of a Tg[ET(krt4:EGFP)sqet4] (green) larval
neuromast labeled with DAPI (red) showing
hair cells (A) before neomycin treatment and
(B) 72 h after the 6th treatment. (C) Image of
a Tg[ET(krt4:EGFP)sqet4] (green) larval
neuromast counterstained for cellular
membranes (red) 24 hpt, with 8 hair cells.
(D) Graph depicting the number of hair cells
per neuromast 24 h after each neomycin
treatment, over the course of 6 consecutive
treatments. (E-F) A neuromast from a
neomycin-treated larva Tg[Atoh1a:dTomato
(red) ; ET(krt4:EGFP)sqet4 (green)] without
(E), and with Notch inhibition with DAPT (F),
showing more numerous hair cells and
stronger and broader Atho1a expression.
(G) Graph showing number of hair cells per
neuromast 24 h after two neomycin
treatments with (green) and without (red)
inhibition of Notch. DAPT incubation
period is shadowed in blue. Results are
mean±s.d. Time points: 0 h N=5 neuromasts
(5 animals), 24 h N=8 neuromasts
(8 animals), 48 h N=4 neuromasts
(4 animals).

neuromast epithelium has self-organizing properties, which may have progress into terminal differentiation are spatially fixed, resulting in
evolved to allow fish to maintain organ proportions during growth the preferential production of hair cells in permissive polar areas.
and repair (Fig. 5). However, a single change in intercellular signaling can route a large
Collectively, the results shown above suggest that pool of sustentacular cells into acquiring a progenitor fate, resulting
compartmentalized Notch signaling determines the geometric order in the ectopic production of supernumerary hair cells. The resulting
of hair-cell regeneration. Thus, it will be important to discover what imbalance of tissue proportions is reversible, however, supporting
controls the spatiotemporal activation of the Notch receptor. One the notion that distinct neuromast regions are territories, as opposed
possibility is that polarized activating signals emanate from the to compartments, because they contain plastic cellular identities that
hair cells. This could also explain the seemingly contradictory are dynamically maintained by continuous intercellular signaling. In
observations that although the expression of a Notch receptor is up- that, the neuromast is more reminiscent to the Drosophila intestine,
regulated in broad areas of the neuromast epithelium during the first where Notch signaling generates plastic stem-cell niches that
24 h after hair-cell ablation, it becomes co-expressed with Atoh1a and promote localized regeneration (Mathur et al., 2010). In sum, here
does not repress hair-cell production (Ma et al., 2008). Analyzing we present evidence that epithelial self-organization underlies the
Biology Open

neuromasts chronically devoid of hair cells will clarify this issue. non-deterministic acquisition of UHCP fate, which governs the
inexhaustible hair-cell regeneration of the lateral line.
CONCLUSIONS
During organ repair, the number and spatial distribution of different MATERIALS AND METHODS
cells must be tightly coordinated. Similarly to the Drosophila ovary DNA constructs
and the mammalian skin (Blanpain and Fuchs, 2014; Xie and The plasmid Cldnb::ERT2-GAL4 that was used to generate the stable
Spradling, 2000), neuromast regions that allow progenitor cells to transgenic line was placed in a miniTol2 backbone and was a gift of

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Fig. 4. Neuromast organization after recurrent damage. (A-F) A neuromast immunostained for Sox-2 (red) and labeled with DAPI (blue) in (A-C) controls
and (D-F) after induction of constitutive Notch signaling. (G) Quantification of Sox-2 positive cells and total cells number in regenerating neuromast 24 h
after neomycin treatment with and without induction of NICD in the double transgenic line Tg[Cldnb:Gal4ERT2;5xUAS-E1b:6xMYC-notch1a], results are
mean±s.e.m. (Mann-Whitney test U=110, Control N=16 (3 larvae) and NIND=23 (4 larvae), *P=0.03). (H) Quantification of regenerated hair cells 24 hpt after
constitutive Notch activation by heat shock and chemical induction, results are mean±s.e.m. (Control versus Heat shock Mann-Whitney U=7.5, P<0.0001.
Biology Open

Control versus Induction Mann-Whitney U=0, P<0.0001. Control N=33 (8 larvae), Heat shock N=7 (1 larvae), Induction N=14 (3 larvae) (I) Quantification of
sustentacular, hair and mantle cells in regenerating neuromasts after two consecutive neomycin treatments, intercalated with a DAPT incubation period, results
are mean±s.e.m. [Sust. cells Mann-Whitney U=61.5, P=0.0052. Hair cells Mann-Whitney U=34.5, P<0.0001. Mantle cells Mann-Whitney U=135, P=0.8081.
Control N=22 (4 larvae), DAPT N=15 (3 larvae)]. (J-K) Representative neuromasts of the triple transgenic line Tg[ET(krt4:EGFP)sqgw57A ; Alpl:mCherry ; pou4f3:
Kusabira-CAAX] used for quantifications. (L) Graph with hair-cell counts in regenerating neuromasts of Tg[ET(krt4:EGFP)sqet4] fish during 7 days, showing
controls (red) and DAPT-treated fish (green). DAPT incubation period is indicated by the blue vertical bar. Results are mean±s.d. Time points: 0 h N=4
neuromasts (4 animals), 24 h N=5 neuromasts (5 animals), 182 h N=9 neuromasts (9 animals).

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D. Wilkinson (NIRM, UK). To generate Tg[Cldnb:Gal4ERT2] line, Pharmacology

20 pg of the Tol2-expression clone and 20 pg of the transposase Neomycin treatments were performed on 5–6 days post fertilization (dpf )
synthetic RNA were simultaneously injected into one-cell stage transgenic zebrafish larvae or 6- to 2-year old adult fish. Neomycin
wild-type eggs. The resulting embryos were raised to adulthood and sulfate at 10 mg/ml stock solution in dH2O (Sigma, St. Louis, MO USA),
out-crossed for visual screening of germ-line transmission of the was diluted in system water to a final concentration of 250 µM. Larvae
transgene. were incubated with neomycin solution for 1 h at RT, rinsed and allowed
to recover in system water at 28.5°C. Sibling fish in system water at RT
Zebrafish strains and husbandry for 1 h and then transferred at 28.5°C served as control. Notch signaling
Zebrafish were maintained under standardized conditions and experiments was inhibited using the γ-secretase inhibitor DAPT (N-[N-(3,5-
were conducted in accordance with protocols approved by the Ethical difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester) (Sigma).
Committee of Animal Experimentation of the Parc de Recerca Biomèdica de DAPT was reconstituted in 10% DMSO to a stock concentration of
Barcelona (PRBB), Spain. The ET(krt4:EGFP)sqgw57A line was generated 150 mM and then diluted to a final concentration of 50 μM solution
by random integration of an gene-trap construct (Kondrychyn et al., 2011). of DAPT in system water with 1% DMSO. Water with 1% DMSO served
The following transgenic lines were described previously: Tg[ET(krt4: as control. For chemical induction of Gal4, 4-hydroxy-tamoxifen (H7904,
EGFP)sqet4], Tg[ET(krt4:EGFP)sqet20] (Parinov et al., 2004), Tg[Cldnb: Sigma) was dissolved at 12.5 mg/ml in 100% ethanol and stored at
mem-EGFP] (Haas and Gilmour, 2006), Tg[ pou4f3:gap43-GFP] (Xiao −20°C. Subsequent dilutions in embryo medium were made prior to use
et al., 2005), Tg[hsp70l:Gal4], Tg[5xUAS-E1b:6xMYC-notch1a] (Scheer to a final concentration of 10 μM. Control larvae were incubated with an
et al., 2001), Tg[myo6b:actb1-EGFP] (Kindt et al., 2012), and Tg[Alpl: equivalent amount of ethanol diluted in embryo medium.
mCherry] (Steiner et al., 2014), Tg[ pou4f3:Ribeye-Kusabira] (Pujol-Martí
et al., 2014). Vital imaging of zebrafish larva
For vital imaging, zebrafish larvae at 6 dpf were anesthetized with a 610 µM
Heat-shock induction of gene expression solution of the anaesthetic 3-aminobenzoic acid ethyl ester (MS-222).
Transgenic fish carrying the hsp70 promoter were incubated at the desired Mechanoreceptive hair cells were identified by labeling with the vital dye
developmental stage at 39°C for 30 min in groups of 30–40 embryos in 2 ml Di-2-ASP (Molecular Probes, Eugene, OR USA) or by EGFP expression in
Eppendorf tubes containing a total volume of 1 ml embryo medium transgenic fish. Samples were mounted onto a glass-bottom 3 cm Petri dish
(Danieau 30%). Upon incubation embryos were returned to petri dishes (MatTek, Ashland, MA USA) and covered with 1% low-melting-point
containing embryo medium fish medium at 28.5°C where they recovered agarose with diluted anaesthetic. Images were acquired with an inverted
until the required time point. confocal microscope with a 40× air or 63× water-immersion objective
lenses.

Imaging adult caudal fins

For imaging caudal-fin neuromasts, adult zebrafish were anesthetized with a
610 µM solution of MS-222 and caudal fins were cut with a sharp scalpel.
Fish were quickly returned to a tank with system water and allowed to fully
recover before being transferred to a running-water system. Cut fins were
fixed at 4°C in a solution of 4% paraformaldehyde (PFA) in phosphate-
buffered saline (PBS) containing 0.2% Tween-20 (PBST). After fixation,
the fins were washed in the same solution without fixative and transferred to
a solution of Vectashield with DAPI (Vector Laboratories, Peterborough,
UK) for at least one hour before imaging.

Immunohistochemistry
Immunohistochemistry was done as described previously (Pujol-Martí
et al., 2012). In brief, samples were fixed overnight at 4°C in a solution of
4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS)
containing 0.2% Tween-20. After fixation, the samples were washed
with PBS containing 1% Tween-20 and permeabilized by incubation in
acetone at −20°C for 8 min. Samples were washed with PBS 1% tween-
20 and blocked at room temperature with 10% bovine serum albumin.
Primary antibody was incubated for 48 h at 4°C in PBS with 0.2%
Tween-20 and secondary antibody was incubated overnight at 4°C in
PBS with 0.2% Tween-20. Antibodies were used at the following
dilutions: rabbit anti-Sox-2 (AbCam, Cambridge, UK) at 1:200. Texas
Red-labeled donkey anti-rabbit immunoglobin secondary antibody
(Molecular Probes, Life Technologies, Paisley, UK) and Alexa Fluor®
488 goat anti-rabbit immunoglobin secondary antibodies (A5040, Sigma)
at 1:500. Images were obtained using a confocal microscope (LSM 510;
Carl Zeiss).
Biology Open

Quantification of hair cells

To quantify hair cells, Tg[ET(krt4:EGFP)sqet4] zebrafish larvae were used.
Confocal stacks of neuromasts were acquired using an LSM 510 Zeiss
microscope. Hair cells were manually identified by expression of
Fig. 5. Model of neuromast self-organization. Schematic representation of cytoplasmic GFP. All data was processed and analyzed using a non-
the current model of epithelial self-organization during hair-cell regeneration, parametric test Mann-Whitney U-test (P<0.05, two-tailed) with GraphPad
depicting the status of supporting cells upon Notch inhibition and after Prism version 6.04 for Windows (GraphPad Software, La Jolla, CA USA,
recovery. www.graphpad.com).

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Acknowledgements Kindt, K. S., Finch, G. and Nicolson, T. (2012). Kinocilia mediate

We are indebted to S.-i. Higashijima, D. Gilmour, L. Godinho, V. Korzh, A. Steiner, mechanosensitivity in developing zebrafish hair cells. Dev. Cell 23, 329-341.
A. J. Hudspeth, H. Baier, S. Gerety and the EZRC for reagents and zebrafish strains, Kondrychyn, I., Teh, C., Garcia-Lecea, M., Guan, Y., Kang, A. and Korzh, V.
and to the animal facility of the PRBB (Barcelona, Spain) for animal care. (2011). Zebrafish Enhancer TRAP transgenic line database ZETRAP 2.0.
Zebrafish 8, 181.
Langer, T., am Zehnhoff-Dinnesen, A., Radtke, S., Meitert, J. and Zolk, O.
Competing interests
(2013). Understanding platinum-induced ototoxicity. Trends Pharmacol. Sci. 34,
The authors declare no competing or financial interests.
458-469.
Ló pez-Schier, H. and Hudspeth, A. J. (2006). A two-step mechanism underlies the
Author contributions planar polarization of regenerating sensory hair cells. Proc. Natl. Acad. Sci. USA
H.L.-S. designed the project and analyzed the data together with F.P.-T. and O.V.-L. 103, 18615.
F.P.-T., O.V.-L., L.P.-M., M.T., E.G.-G. and E.T.-M. performed experiments. O.V.-L., Lush, M. E. and Piotrowski, T. (2014). Sensory hair cell regeneration in the
F.P.-T. and H.L.-S. wrote the paper, which was read and approved by all the authors. zebrafish lateral line. Dev. Dyn. 243, 1187-1202.
Ma, E. Y., Rubel, E. W. and Raible, D. W. (2008). Notch signaling regulates the
Funding extent of hair cell regeneration in the zebrafish lateral line. J. Neurosci. 28,
This work was funded by the FCT of Portugal through the GABBA PhD program to 2261-2273.
Mathur, D., Bost, A., Driver, I. and Ohlstein, B. (2010). A transient niche regulates
F.P.-T., and by a grant from the European Research Council (ERC-StG) to H.L.-S.
the specification of Drosophila intestinal stem cells. Science 327, 210-213.
Millimaki, B. B., Sweet, E. M. and Riley, B. B. (2010). Sox2 is required for
Supplementary material maintenance and regeneration, but not initial development, of hair cells in the
Supplementary material available online at zebrafish inner ear. Dev. Biol. 338, 262-269.
http://bio.biologists.org/lookup/suppl/doi:10.1242/bio.012112/-/DC1 Neves, J., Vachkov, I. and Giraldez, F. (2013). Sox2 regulation of hair cell
development: incoherence makes sense. Hear. Res. 297, 20-29.
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