ABSTRACT Hearing loss is a serious burden to physical and mental health worldwide. Aberrant development and damage of hearing organs are recognized as the causes of hearing loss, the

molecular mechanisms underlining these pathological processes remain elusive. Investigation of new molecular mechanisms involved in proliferation, differentiation, migration and maintenance

of neuromast primordium and hair cells will contribute to better understanding of hearing loss pathology. This knowledge will enable the development of protective agents and mechanism study

of drug ototoxicity. In this study, we demonstrate that the zebrafish gene _miles-apart_, a homolog of sphingosine-1-phosphate receptor 2 (s1pr2) in mammals, has an important role in the

regulation. Both up- and downregulation of _miles-apart_ led to abnormal otoliths and semicircular canals, excess or few hair cells and neuromasts, and their disarranged depositions in the

lateral lines. Miles-apart (Mil) dysregulation also caused abnormal expression of hearing-associated genes, including _hmx2_, _fgf3_, _fgf8a_, _foxi1_, _otop1_, _pax2.1_ and _tmieb_ during

zebrafish organogenesis. Moreover, in larvae _miles-apart_ gene knockdown significantly upregulated proapoptotic gene _zBax2_ and downregulated prosurvival gene _zMcl1b_; in contrast, the

level of _zBax2_ was decreased and of _zMcl1b_ enhanced by _miles-apart_ overexpression. Collectively, Mil activity is linked to organization and number decision of hair cells within a

neuromast, also to deposition of neuromasts and formation of otic vesicle during zebrafish organogenesis. At the larva stage, Mil as an upstream regulator of _bcl-2_ gene family has a role

in protection of hair cells against apoptosis by promoting expression of prosurvival gene _zMcl1b_ and suppressing proapoptotic gene _zBax2_. SIMILAR CONTENT BEING VIEWED BY OTHERS

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LOSS Article Open access 18 November 2022 MAIN Hearing loss not only impairs personal physical and mental health but also increases a social and economic burden. Hearing damage can be

caused by a wide range of factors, which include defects of hair cells and inner ear structures, but the defect molecular targets remain elusive. Thus, identification of new molecular

mechanisms associated with developmental processes of hearing organs such as migration of the neuromast primordium, formation of inner ear, differentiation and maintenance of hair cells, as

well as hair cell apoptosis, will contribute to a better understanding of hearing loss pathology. This knowledge developing will facilitate the development of new medicines to prevent

ototoxicity and protect hearing. Sphingosine-1-phosphate (S1P) is a lipid-active mediator and signal molecule, in addition to being a component of the cell membrane. It is involved in

regulating multiple physiological functions, including cell movement, migration, growth and survival, vasculogenesis, nervous and immune system function, reproduction, lymphocyte transfer

and audition.1, 2, 3 S1P is a ligand for five human G protein-coupled receptors (GPCRs), called S1P1, S1P2, S1P3, S1P4 and S1P5. Dissimilarly, mice possess only three receptors: S1p1, S1p2

and S1p3. Though they are all coupled with G-proteins, the S1P receptors do not demonstrate functional redundancy. Interactions among the receptors are mediated by tyrosine kinase

receptors.4 Transduction of S1P signaling occurs mostly by combining with Edg family receptors.5, 6, 7 Although most reports of S1P receptor function relate to the cardiovascular, nervous

and immune systems,8, 9, 10, 11 there is some evidence that S1P receptors are related to the function of cochlea and vestibular apparatus. Herr _et al._2 found that S1p1, S1p2 and S1p3 are

expressed in mouse cochlea, and that S1p2 levels were higher than those of S1p1 and S1p3, and upregulated along with the developmental process. S1p2-knockout mice had normal cochlear and

vestibular development initially, but rapid degeneration of these structures led to deep deafness by 22 days of age and regressive lost of vestibular function with their age growth.

S1p2/S1p3 double knockout caused increased perinatal lethality and decreased litter size. As in S1p2-knockout mice, significant and progressive degeneration of auditory and vestibular organs

also occurred in S1p2/S1p3 double knockout mice with age, culminating in complete hearing loss.1, 2 These results indicate that S1p receptors have important roles in maintaining hair cell

functions in mice. In zebrafish, mutation of the S1P2 ortholog, miles-apart (Mil), resulted in a cardia bifida phenotype. This defect in cardiac development is due to failed migration of

heart precursor cells to the midline, revealing an important function of the S1P2 receptor in directing cardio-primordium localization and cardiac tube fusion in zebrafish. Loss of Mil

function also caused edematous pericardial sac and epithelium-like blistering in the tail.5, 8, 10 However, there are no reports for zebrafish on the role of Mil in the development of

auditory and vestibular organs, such as hair cells and neuromasts. Zebrafish hair cells are similar to their mammalian counterparts in both morphology and function, and reside inside the

otic vesicles and in the neuromasts at the lateral line system on the body surface. These characteristics make zebrafish a good model system to study the effects of drug toxicity on hearing

and protection and regeneration of hair cells.1, 2 In the zebrafish embryonic stage each otic capsule contains two maculae: the anterior utriculus has a vestibular function, and the

posterior sense macula (saccule) has a role in hearing. Each macula is covered by an otolith, which is attached to a kinocilium of hair cells. Semicircular canals begin to form by 48 h.p.f.

(hour post fertilization). The number of hair cells in maculae and lateral lines increases rapidly from 3 to 7 days post fertilization (d.p.f.), and by 7 d.p.f. all the key structures have

emerged (the lagena, a major auditory macula, does not form until several days later). Neuromasts in the lateral line on the body surface are very similar in structure and function to the

sensory maculae of the inner ear.12 These functions are mainly dependent on the physiological activity of the hair cells. In this study, we explored the functions of zebrafish _miles-apart_

in hair cell and neuromast development and survival and report the first association of _miles-apart_ activity with the development of neuromasts, hair cells and otic vesicles, as well as

with the expression of auditory-related genes and bcl-2 gene family. We infer that Mil may be an important regulator for the formation and maintenance of hearing organs in zebrafish. Our

findings will contribute to a better understanding of the processes of hearing development and facilitate research of otoprotective agents. RESULTS EXPRESSION PATTERN OF _MILES-APART_ DURING

ZEBRAFISH ORGANOGENESIS Whole-mount _in situ_ hybridization was used to detect _miles-apart_ spatiotemporal expression pattern during zebrafish embryonic development. The _miles-apart_

expression was mainly aggregated in the encephalic region, particularly in the midbrain/hindbrain boundary and the ventral hindbrain; and weak expression also was observed in somites but not

in notochord at 24 h.p.f. (Figures 1a, a′ and a′′). At the pharyngula stage (48 h.p.f.), the _miles-apart_ transcription focused on the midbrain/hindbrain boundary, midbrain, ventral

hindbrain area, and in floor plate of the neural tube and vasculature, particularly appeared in a regular and discrete pattern at the positions of the future neuromasts on the lateral lines

(Figures 1b, b′ and b′′). However, at the hatching period (72 h.p.f.), the _miles-apart_ mRNA was not detected in the lateral lines and in the posterior floor plate but was still highly

expressed in the midbrain/hindbrain boundary, the brainstem and the floor plate (Figure 1c). These results suggest that the _miles-apart_ expression pattern is correlated to development of

the nervous system, including the brain, the central nervous tube and the neural sensory system in zebrafish. DYSREGULATION OF _MILES-APART_ EXPRESSION DISTURBS DEVELOPMENT OF HAIR CELLS AND

OTIC VESICLES Using the indication that high _miles-apart_ expression occurred in the ventral forebrain and midbrain, midbrain/hindbrain boundary, brainstem and the lateral line neuromasts

in 48 h.p.f. WT embryos, we hypothesized that Mil functions may be associated with the formation of neuromasts and hair cells beside its function in development of the central nervous

system. We investigated Mil function on development of the otic vesicle in zebrafish embryo using down- and upregulation of _miles-apart_ expression. An antisense morpholino oligo (MOmil)

binding to the _miles-apart_ translation initiation site was used for inhibition of _miles-apart_ translation by injection into embryos at the one- to four-cell stage. The morphant embryos

displayed aberrant otic vesicle and otoliths that utricles became non-oval, disfigure and bigger than saccule; and in contrast, saccule morphous presented small, deficient and non-spherical

structures at 48 h.p.f. (9/10). The size ratio of saccule to utricle changed significantly compared with the WT ratio (Figure 2a). The semicircular canals had defect structure such as a gap

or breakage developed between anterior semicircular canal (psc) and posterior semicircular canal (psc), beside the otolith abnormality in 72 h.p.f. embryos (11/15) (Figure 2b).

Correspondingly, capped mRNA of Mil was injected into embryos at the same stage for _miles-apart_ overexpression. In these embryos, the utricle appeared shrunk and smaller, and saccular

otolith was deformed and divided at 48 h.p.f. (10/10) (Figure 2a). At 72 h.p.f., the middle junction of the semicircular canals was deficient, or even all the semicircular canals totally

absent (7/11) (Figure 2b). These data suggest that _miles-apart_ gene dysregulation could considerably result in mis-patterning of the otic vesicle and abnormal structure of otoliths. Hair

cell staining by YO-PRO-1 was used to examine the impact of _miles-apart_ on the formation and deposition of hair cells and neuromasts in the lateral lines on the surface of zebrafish head

and body. The results show that in the 6 d.p.f. morphants at the posterior lateral line several neuromasts were absent or shrunken (Figure 3b); and at the anterior lateral line number of the

hair cells in neuromast orbital 1 (o1), middle lateral line 1 (ml1), middle lateral line 1 (ml2), orbital 2 (o2) and infraorbital 4 (io4), which resided at the otic vesicles around, were

counted and found that the hair cells were obviously diminished in neuromast o1 and o2 (Figures 3g and j), as opposed to the ordered hair cell aggregation in the WT group and in the

mismatched MO (misMO) control group (Figures 3f and h). However, when _miles-apart_ expression was upregulated by mRNA injection, excess neuromasts in the posterior lateral line and ectopic

and fluffy hair cells in the five neuromasts around the otic vesicle were observed (Figures 3d, e, I and j). Statistical analysis presents a significant difference of the neuromast numbers

between the morphants and mRNA-injected larvae but not between WT larvae and the three injection groups, respectively (Figure 3e). In addition, compared with the WT group the hair cell

number of o1 and o2 in the morphants and of ml1 in the mil overexpressed larvae were changed significantly (Figure 3j). These data suggest that one of Mil functions probably is involved in

controlling both the number and organization of hair cells in a neuromast, and also in controlling the number and localization of neuromasts in the lateral lines. DYSREGULATION OF

_MILES-APART_ AFFECTS EXPRESSION OF HEARING-ASSOCIATED GENES IN ZEBRAFISH EMBRYO In order to explore whether _miles-apart_ expression is correlated with genes that involved in the developing

of hair cells and neuromasts, we examined expression levels of hearing-related genes such as _hmx2_, _fgf3_, _fgf8a_, _foxi1_, _otop1_, _pax2.1_ and _tmieb_, in 48 h.p.f. embryos. This time

point corresponds to the stage while the otic vesicle is developing and the pro-neuromast primordium begins to migrate. The results show that transcription of these genes was reduced in a

concentration-dependent manner in MOmil-injected embryos, and four of them _hmx2_, _fgf3_, _foxi1_ and _pax2.1_ reduced in statistical significance (Figures 4a and c). As injection mock, in

the misMO group these gene levels slightly went up or down, and _hmx2_ and _tmieb_ showed descent significantly (Figures 4b and d). Conversely, these gene levels were elevated in the

mRNA-injected embryos and the elevation of _fgf3_, _foxi1_ and _tmieb_ are in statistical significance (Figures 4e and f). So, we suppose that these genes may be effectors of Mil activity

for the development and functions of hearing organs. SURVIVAL OF HAIR CELLS MAY BE ASSOCIATED WITH _MILES-APART_ FUNCTION BY SUPPRESSING _ZBAX2_ AND PROMOTING _ZMCL1B_ Respecting deficiency

of the hair cells and otic vesicles resulted from MOmil injecton, we asked whether _miles-apart_ knockdown prompted hair cell death and which mechanism was involved in? Survival of the larva

hair cells was detected using an apoptosis staining kit (Invitrogen, Carlsbad, CA, USA) with the three dyes. Hair cells in neuromast ml1 and o2 were counted and analyzed by statistics. The

results show that compared with WT hair cells, number of live hair cells in 6 d.p.f. morphants decreased with dimly stained by Hoechst33324, the apoptotic hair cells with condensed chromatin

increased by stained brightly by Hoechst33324 and green fluorescence by YO-PRO-1 dye, and the dead hair cells also grew up with primarily red fluorescence and some green fluorescence in a

concentration-dependent manner (Figure 5). Meanwhile, several apoptosis/prosurvival genes including bcl-2 family and caspase family were detected using qPCR. Significantly, _zMcl1b_, a

prosurvival gene was downregulated and a proapoptotic gene, _zBax2_, was upregulated in the morphants. In contrast, significantly too, _zMcl1b_ was increased and _zBax2_ decreased in the

larva with mil-mRNA injection (Figure 6). So, we suppose that _miles-apart_ knockdown may have sensitized the hair cell to apoptosis by enhancing _zBax2_ expression and suppressing _zMcl1b_

expression. One of Mil functions is probably involved in hair cell survival. DISCUSSION The inner ear and lateral line systems are important organs for hearing and sensing fluid flow or

pressure waves associated with the approach of other animals in vertebrates. The development of inner ear and lateral line system is involve in spatiotemporal patterning of

hearing-associated gene expression, expansion of the otic vesicle signaling cascade, cell migration and interaction with neighboring tissues.13, 14 Previous studies of knockout mice showed

that the loss of S1p2 disturbed mouse hearing, thus concluding that the S1p2 has an important physiological role for survival and functional maintenance of hair cells in mice.1, 2 However,

the role of the zebrafish S1p2 ortholog, Mil, in hair cell function remains to be investigated. In this study, we observed _miles-apart_ expression in the encephalic region including the

hindbrain where the otic capsule resides, and at the typical neuromast preset positions in the lateral lines of 48 h.p.f. WT embryos. This is likelihood to predict the future neuromast

deposition in the lateral lines. Between 48 and 72 h.p.f., _miles-apart_ expression disappeared from the embryonic trunk, suggesting that _miles-apart_ is subjected to rigorous

spatiotemporal regulation for the lateral line system. Using MOmil-mediated gene knockdown and _miles-apart_ capped-mRNA overexpression, a role for Mil in organization and maintenance of

hair cells, neuromasts and otic vesicle organ was identified. Further, we explored potential _miles-apart_ targeting genes that are known to be associated with the development and function

of the otic vesicle and hair cells. Seven hearing-related genes were found to be coincidentally regulated by _miles-apart_ level variation. They were upregulated, whereas Mil was

overexpressed and reduced when _miles-apart_ was knocked down. Thus, we infer that Mil can be an important upstream regulator to the seven genes. In view of the literatures, _fgf3_ and

_fgf8_ are required for inner ear and hindbrain patterning, induction of otic placode and migration of PLL primordia.14, 15, 16, 17 _foxi1_ is the earliest marker for otic precursor cells

and otic placode formation,18 whereas _pax2.1_ were essential for the induction of functional hearing cells in both the inner ear and the lateral lines.19, 20 Notably, _pax2.1_ is

specifically expressed in hair cells during differentiation, and is also maintained at high levels in mature hair cells, but is not expressed in support cells.19 _hmx2_ is involved in the

formation of mechanosensory neuromasts,21 and _otop1_ was identified as being required for otolith initiation and development of otolith size and shape.16, 22 And _tmieb_ was identified as

being involved in the functioning of both auditory and vestibular mechanoelectrical transduction.23 These data support the inference in this study that _miles-apart_ gene activity is likely

linked to hearing organ development and is probably mediated by the above hearing-associated genes. Previous research showed that Mil was responsible for directing migration and fusion of

cardiac precursor cells at the heart field and for subsequent differentiation into a functional organ in cardiovascular development.5, 8, 24 In the present study, we find that Mil function

may be partly involved in migration and development of hair cell precursors and pro-neuromast primodia in ALL and PLL (Figure 3). The functional concordance of Mil in divergent organs

suggests a basic common role of Mil in directing precursor cell migrating and positioning in zebrafish organogenesis. Our results also show that knockdown of _miles-apart_ gene increased the

number of apoptotic hair cells and dead hair cells, decreased hair cell survival and caused otic vesicle structure damaged. These phenomena are similar to that of cochlear and vestibular

degeneration in S1p2-knockout mice.2 Moreover, the significant results that proapoptotic gene _zBax2_ and prosurvival gene _zMcl1b_ can be tightly regulated by Mil indicate that Mil may be

an upstream coregulator for _zBax2_ and _zMcl1b_ gene expression. As we know, zMcl and zBax2 interactions regulate the survival/apoptosis pathway in zebrafish. Kratz _et al._25 reported that

zMcl-1a and zMcl1b function as gatekeepers against apoptosis induction by stimulation of the extrinsic pathway. Knockdown of _zMcl-1a_ and _zMcl-1b_ decreases viability of zebrafish embryos

and sensitizes zebrafish embryos to apoptosis induction. zBax2 was homologous to mammalian Bax in sequence and synteny, but functional conservation with human Bak. Coincidentally, in human

healthy cells, Bak keeps a silent proapoptotic role by associating with Mcl-1 and Bcl-x(L); releasing of Bak can activate apoptosis process.26 On the basis of these literatures and our

current results, we propose a model of Mil mechanism underlining the development and survival of hair cells and neuromast of the lateral lines in zebrafish. During the embryonic

organogenesis stage, Mil as a position signal guides the migration and deposition of pro-neuromast primodia at correct positions in ALL and PLL; Mil knockdown led to deficient deposition

signals and reduced neuromasts at the lateral lines, which just as a Mil mutant failed to direct cardiomyocytes to migrate to the midline.5 Meanwhile, Mil also participates in activating the

expression of hearing genes that subsequently induce development of the otic vesicles and differentiation of pro-neuromast primodia into neuromast and hair cells. Since 2 days post

hatching, zebrafish larvae begin to live independently with some behavioral capacity including swim, hiding and looking for food, which means their functional balance and hearing emerged.

Execution of these functions is guaranteed by enough normal hair cells and the normal lateral lines. Mil, probably as an upstream regulator of _bcl-2_ gene family, protects hair cells

against apoptosis by upregulating expression of prosurvival gene _zMcl1b_ and downregulating proapoptotic gene _zBax2_. Knockdown of _miles-apart_ gene can produce excess zBAX2 and deficient

zMCL1B, which leads to plenty of free zBAX2 that sensitizes hair cells to apoptosis. This study first provides evidence that _miles-apart_ gene function is involved in development of the

otic vesicles and the lateral line of hair cells and neuromasts in zebrafish, and that change of the Mil level can regulate expression of bcl-2 family gene _zBax2_ and _Mcl1B_. MATERIALS AND

METHODS ETHICS STATEMENT This study was carried out in strict accordance with the recommendations in the Regulation for the Management of Laboratory Animals of the Ministry of Science and

Technology of China. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences

(IMBF20060302). ZEBRAFISH MAINTENANCE AND EMBRYO COLLECTION Zebrafish (_Danio rerio_) WT Tuebingen strain was originally obtained from the College of Life Sciences, Beijing University. The

fish were raised at 28.5±1 °C in a 14/10 h light–dark cycle.27 Embryos were obtained by natural mating; synchronous embryos at the appropriate stage were collected and recorded by h.p.f. and

d.p.f.28 Embryos were fixed with 4% paraformaldehyde in phosphate-buffered saline for _in situ_ hybridization or immersed in Trizol reagent (Invitrogen) for mRNA isolation. Pigmentation in

embryos past 24 h.p.f. was prevented by incubating in phenylthiourea (PTU) (Sigma-Aldrich, Saint Louis, MO, USA) before being fixed. SYNTHESIS OF MOS AND _MILES-APART_ CAPPED-MRNA Antisense

morpholino oligonucleotides of MOmil and misMO were designed and bought from Genetools (Philomath, OR, USA; http://www.gene-tools.com). MOmil was used for the inhibition of _miles-apart_

translation by binding to _miles-apart_ initiation sites, with the sequence 5′-CCGCAAACAGACGGCAAGTAGTCAT-3′. The control misMO’ was designed for negative injection control in which five

bases were missense and labeled with underline in sequence 5′-CCCCAAACACACCGCAACTACTCAT-3′. Both MOs were redissolved at a stock solution of 1.0 _μ_mol/ml in ddH2O. The full-length

_miles-apart_ cDNA sequence was cloned from 24 h.p.f. WT embryos using Reverse Transcriptase polymerase chain reaction (RT-PCR) and primers designed according to the zebrafish _miles-apart_

sequence logged in GenBank (NM_001159970), and was identified by sequencing (Sangon Biotech, Shanghai, China). This cDNA was used as a template for _miles-apart_ capped mRNA synthesis using

an Ambion mMESSAGE mMACHINE T3 kit (Applied Biosystems, Foster City, CA, USA), and the product was tested for quality and yield by electrophoresis. MICROINJECTION For hair cell staining, 1 

nl of 20 _μ_M MOmil or 20 ng/_μ_l _miles-apart_ capped mRNA was separately injected into WT embryos at one- to four-cell stage; at least five larvae at 6 d.p.f. were used for counting of

hair cells of the neuromasts o1, o2, ml1, ml2 and io4 and for counting of neuromasts at the post lateral lines. For otic capsule observation of living larvae and counting of hair cells, 1 nl

of 20 _μ_M MOmil or 20 ng/_μ_l _miles-apart_ capped mRNA were injected into embryos at the same stage. For apoptosis staining of hair cells, 1 nl of MOmil at 40 _μ_M and 500 _μ_M, and misMO

at the same concentrations, were respectively injected into one- to four-cell embryos; then the larvae were collected at 6 d.p.f. For RT-PCR analysis of hearing-related genes, 1 nl of 10,

20 or 40 _μ_M MOmil, and 10, 20 or 40 ng/_μ_l _miles-apart_ capped mRNA were injected at the same embryonic stage. The embryos were incubated and collected at 48 h.p.f. For qPCR analysis of

zBax2 and zMcl1b, 1 nl of 40 _μ_M MOmil and 50 ng/_μ_l _miles-apart_ capped mRNA were separately injected at the same embryonic stage. The embryos were incubated at the same condition above.

OBSERVATION OF OTIC VESICLES The morphous and structure of otic vesicles in WT, MOmil- and _miles-apart_ mRNA-injected zebrafish embryos at 48 and 72 h.p.f. were observed under a

differential interference contrast IX71 microscope (Olympus, Tokyo, Japan) and recorded by a 500D digital camera (Canon, Tokyo, Japan). Each group has more than 10 larvae for otolith

counting and histogram. WHOLE-MOUNT _IN SITU_ HYBRIDIZATION For _in vitro_ synthesis of the RNA probe, _miles-apart_ gene sequence was inserted into pBluescript KS (Agilent, Santa Clara, CA,

USA) then used as a template for _miles-apart_ sense and antisense RNA probe synthesis using a DIG RNA labeling kit (Roche Applied Sciences, Basel, Switzerland). For determination of WT

_miles-apart_ expression patterns, embryos were collected at 24, 48 and 72 h.p.f. Embryos treated with MOmil or mRNA injections, as well as WT controls, were collected at 48 h.p.f. Embryos

allowed to develop past 24 h.p.f. were pretreated with PTU to suppress pigmentation before fixation in 4% paraformaldehyde overnight at 4 °C. Embryos were treated by DNase before

hybridization to remove pseudo-positive interference caused by genomic DNA. Subsequent steps of whole-mount _in situ_ hybridization procedure followed a standard protocol.29 HAIR CELL

STAINING FOR COUNTING AND APOPTOSIS/DEATH DETECTION For hair cell counting, zebrafish larvae at 6 d.p.f. were anesthetized in 0.016% Tricaine (Sigma-Aldrich) and incubated in the fluorescent

dye YO-PRO-1 (Invitrogen) at final concentration 1 _μ_mol/l was used for staining of hair cell nuclei30 of the five neuromasts, o1, ml1, ml2, o2 and io431 which resided at the surface of

the otic vesicle around. The larvae were then visualized using an IX51 inverted phase contrast fluorescence microscope (Olympus). Each group has five larvae for staining and hair-cell

counting. Photographs were taken with a Canon 500D Camera. For apoptosis/death detection of the hair cells, an apoptosis staining kit (Chromatin Condensation/Membrane Permeability/Dead Cell

Apoptosis Kit with Hoechst 33342/YO-PRO-1 and PI for Flow Cytometry. Catalog no. V23201; Invitrogen) was used to inspect whether _miles-apart_ gene knockdown can cause hair cell apoptosis.

The dye mix solution with the three dyes was prepared in the fish water by following the kit instruction. The living larvae at 6 d.p.f. were soaked in the mixed dye solution and wrapped into

tin foil to avoid of light at 28 °C for 30 min, then anesthetized and observed under fluorescence inverted microscope. The five neuromasts, o1, ml1, ml2, o2 and io4 were checked and hair

cells in ml1 and o2 were counted and shot. Then, the dyed hair cells were classified based on the kit instruction: ‘Blue-fluorescent Hoechst33324 brightly stains the condensed chromatin of

apoptotic cells and more dimly stains the normal chromatin of live cells; Green-fluorescent YO-PRO-1 dye can enter apoptotic cells with intact plasma membrane, whereas red-fluorescent

propidium iodide (PI) cannot. Thus, after staining with YO-PRO-1 dye and PI, apoptotic cells show green fluorescence and dead cells show primarily red fluorescence and some green

fluorescence’ (Invitrogen, Catalog no. V23201).32 RT-PCR AND QPCR Embryos or larvae at selected stages were collected into Trizol Reagent. Total RNA was extracted following the Trizol

Reagent RNA extraction kit manual. First-strand cDNAs were synthesized by reverse transcription using the M-MLV RTase cDNA Synthesis Kit (Takara Bio, Shiga, Japan). For the RT-PCR

amplification, 0.5 _l_L of cDNA was used for each reaction with different primer pairs (primer sequences are listed in Table 1). _β_-Actin was amplified as a template loading control. PCR

parameters were 94 °C 60 s, 55 °C 50 s, 72 °C 60 s, for 30 cycles for hearing-related genes, 26 cycles for _miles-apart_ and 25 cycles for _β_-actin. A final incubation at 72 °C for 10 min

was included to ensure complete synthesis. RT-PCR products were subjected to agarose gel electrophoresis. For statistical analysis of density, all results were normalized to _β_-actin and

then to WT. For detection of apoptosis gene _zBax2_ and prosurvival gene _zMcl1b_, the larvae at 6 d.p.f. (for MO and misMO injection groups) and at 4 d.p.f. (for mRNA injection group) were

collected into Trizol reagent; the different collecting time chosen was based on our preliminary experiments. Transcription levels of _zBax2_ and _zMcl1b_ were examined by qPCR (Agilent

Technologies Stratagene Mx3000P, Santa Clara, CA, USA) and parameters were 95 °C 5 min, 95 °C 30 s, 55 °C 30 s, 72 °C 60 s, for 40 cycles. Then the ct values were analyzed by statistics

calculation. STATISTICAL ANALYSIS Statistical analyses for all the data (means±S.D.) were performed using One way ANOVA tests and _P_-values, 0.05 were assigned as significant. Data with

bars in histograms represent means and S.D.’s that are derived from at least triplicates. ABBREVIATIONS * S1P: Sphingosine-1-phosphate * WT: wild-type * d.p.f.: day post fertilization *

h.p.f.: hour post fertilization * MOmil: morpholino oligo * Mil: miles-apart * o1: orbital 1 * ml1: middle lateral line 1 * ml2: middle lateral line 2 * o2: orbital 2 * io4: infraorbital 4

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anemia virus infection _in vitro_. _Virol J_ 2009; 6: 91. Article  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Mr Weixian Wang for his supporting in microinjection, and

thank professor Bo Zhang (Beijing University) provided zebrafish TU stain seedling. This study was supported by The National Natural Science Foundation of China (No. 30772681 and 81173043),

the Chinese National Key Technology R&D Program (2012BAK25B01) and the Presidential Foundation for the governmental public welfare scientific research institutions (No. IMBF 201101).

AUTHOR INFORMATION Author notes * Z-y Hu and Q-y Zhang: Co-first authors. AUTHORS AND AFFILIATIONS * Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking

Union Medical College, Beijing, China Z-y Hu, Q-y Zhang, W Qin, J-w Tong, Q Zhao, Y Han, J Meng & J-p Zhang Authors * Z-y Hu View author publications You can also search for this author

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COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by A Stephanou RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons

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CITE THIS ARTICLE Hu, Zy., Zhang, Qy., Qin, W. _et al._ Gene _miles-apart_ is required for formation of otic vesicle and hair cells in zebrafish. _Cell Death Dis_ 4, e900 (2013).

https://doi.org/10.1038/cddis.2013.432 Download citation * Received: 03 June 2013 * Revised: 30 September 2013 * Accepted: 30 September 2013 * Published: 31 October 2013 * Issue Date:

October 2013 * DOI: https://doi.org/10.1038/cddis.2013.432 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a

shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * miles-apart * hair cells *

neuromast * otoliths * hearing-associated genes * apoptosis