Mitochondrial redox system: A key target of antioxidant therapy to prevent acquired sensorineural hearing loss.

Noise (noise-induced hearing loss), and ototoxic drugs (drug-induced ototoxicity), and aging (age-related hearing loss) are the major environmental factors that lead to acquired sensorineural hearing loss. So far, there have been numerous efforts to develop protective or therapeutic agents for acquired hearing loss by investigating the pathological mechanisms of each types of hearing loss, especially in cochlear hair cells and auditory nerves. Although there is still a lack of information on the underlying mechanisms of redox homeostasis and molecular redox networks in hair cells, an imbalance in mitochondrial reactive oxygen species (ROS) levels that enhance oxidative stress has been suggested as a key pathological factor eventually causing acquired sensorineural hearing loss. Thus, various types of antioxidants have been investigated for their abilities to support auditory cells in maintenance of the hearing function against ototoxic stimuli. In this review, we will discuss the scientific possibility of developing drugs that target particular key elements of the mitochondrial redox network in prevention or treatment of noise- and ototoxic drug-induced hearing loss.

Berberine chloride protects cochlear hair cells from aminoglycoside-induced ototoxicity by reducing the accumulation of mitochondrial reactive oxygen species.

Aminoglycoside, a medicinal category of antibiotics, are used in treatment of Gram-negative bacterial infections. Although they are the most widely-used antibiotics due to their high efficacy and low cost, several main adverse effects have been reported including nephrotoxicity and ototoxicity. Since drug-induced ototoxicity is one of the major etiological causes of acquired hearing loss, we examined cochlear hair cell damages caused by three aminoglycosides (amikacin, kanamycin, and gentamicin), and investigated protective property of an isoquinoline-type alkaloid, Berberine chloride (BC). Berberine, a well-known bioactive compound found from medicinal plants, has been known to have anti-inflammatory, antimicrobial effects. To determine protective effect of BC in aminoglycoside-induced ototoxicity, hair cell damages in aminoglycoside- and/or BC-treated hair cells using ex vivo organotypic culture system of mouse cochlea. Mitochondrial ROS levels and depolarization of mitochondrial membrane potential were analyzed, and TUNEL assay and immunostaining of cleaved caspase-3 were performed to detect apoptosis signals. As the results, it was found that BC significantly prevented aminoglycoside-induced hair cell loss and stereocilia degeneration by inhibiting excessive accumulation of mitochondrial ROS and subsequent loss of mitochondrial membrane potential. It eventually inhibited DNA fragmentation and caspase-3 activation, which were significant for all three aminoglycosides. This study is the first report suggested the preventative effect of BC against aminoglycoside-induced ototoxicity. Our data also suggests a possibility that BC has the potential to exert a protective effect against ototoxicity caused by various ototoxic drugs leading to cellular oxidative stress, not limited to aminoglycoside antibiotics.

Identification of novel missense mutation related with non-syndromic sensorineural deafness, DFNA11 in korean family by NGS.

BACKGOUND: Hereditary hearing loss is one of the most common genetically heterogeneous defects in human. About 70% of hereditary hearing loss is defined as non-syndromic hearing loss showing loss of hearing ability without any other symptoms. Up to date, the identified genes associated with non-syndromic hearing loss are 128, including 52 genes for DFNA and 76 genes for DFNB. Because of high levels of heterogeneity, it is difficult to identify the causative factors for hearing loss using Sanger sequencing. OBJECTIVE: Our aim was to detect causative factors and investigate pathogenic mutations, which co-segregates within the candidate family. METHODS: We used Next Generation Sequencing technique to investigate whole-exome sequences of a Korean family with non-syndromic hereditary hearing loss. The family showed autosomal dominant inheritance pattern. RESULTS: We identified a novel missense variation, c.1978G > A in MYO7A gene, in the family with the autosomal dominant inheritance pattern. c.1978G > A produced Gly660Arg in the motor head domain of Myosin VIIA disrupt the ATP- and actin-binding motif function. CONCLUSION: This study is the first to report pathogenic mutations within MYO7A gene in Korean family and our data would facilitate diagnosing the primary cause of hereditary hearing loss in Korean.

Protective effect of berberine chloride against cisplatin-induced ototoxicity.

BACKGROUND: Cisplatin (CP) is an effective anticancer drug broadly used for various types of cancers, but it has shown ototoxicity that results from oxidative stress. Berberine has been reported for its anti-oxidative stress suggesting its therapeutic potential for many diseases such as colitis, diabetes, and vascular dementia. OBJECTIVE: Organ of Corti of postnatal day 3 mouse cochlear explants were used to compare hair cells after the treatment with cisplatin alone or with berberine chloride (BC) followed by CP. METHODS: We investigated the potential of the anti-oxidative effect of BC against the cisplatin-induced ototoxicity. We observed a reduced aberrant bundle of stereocilia in hair cells in CP with BC pre-treated group. Caspase-3 immunofluorescence and TUNEL assay supported the hypothesis that BC attenuates the apoptotic signals induced by CP. Reactive oxygen species level in the mitochondria were investigated by MitoSOX Red staining and the mitochondrial membrane potentials were compared by JC-1 assay. RESULTS: BC decreased ROS generation with preserved mitochondrial membrane potentials in mitochondria as well as reduced DNA fragmentation in hair cells. In summary, our data indicate that BC might act as antioxidant against CP by reducing the stress in mitochondria resulting in cell survival. CONCLUSION: Our result suggests the therapeutic potential of BC for prevention of the detrimental effect of CP-induced ototoxicity.

Fursultiamine Prevents Drug-Induced Ototoxicity by Reducing Accumulation of Reactive Oxygen Species in Mouse Cochlea.

Drug-induced hearing loss is a major type of acquired sensorineural hearing loss. Cisplatin and aminoglycoside antibiotics have been known to cause ototoxicity, and excessive accumulation of intracellular reactive oxygen species (ROS) are suggested as the common major pathology of cisplatin- and aminoglycoside antibiotics-induced ototoxicity. Fursultiamine, also called thiamine tetrahydrofurfuryl disulfide, is a thiamine disulfide derivative that may have antioxidant effects. To evaluate whether fursultiamine can prevent cisplatin- and kanamycin-induced ototoxicity, we investigated their preventive potential using mouse cochlear explant culture system. Immunofluorescence staining of mouse cochlear hair cells showed that fursultiamine pretreatment reduced cisplatin- and kanamycin-induced damage to both inner and outer hair cells. Fursultiamine attenuated mitochondrial ROS accumulation as evidenced by MitoSOX Red staining and restored mitochondrial membrane potential in a JC-1 assay. In addition, fursultiamine pretreatment reduced active caspase-3 and TUNEL signals after cisplatin or kanamycin treatment, indicating that fursultiamine decreased apoptotic hair cell death. This study is the first to show a protective effect of fursultiamine against cisplatin- and aminoglycoside antibiotics-induced ototoxicity. Our results suggest that fursultiamine could act as an antioxidant and anti-apoptotic agent against mitochondrial oxidative stress.in cochlear hair cells.

KL1333, a derivative of ß-lapachone, protects against cisplatin-induced ototoxicity in mouse cochlear cultures.

Cisplatin (CP) is a chemotherapeutic drug used to treat cancerous solid tumors, but it causes serious side effects, including ototoxicity. The major cause of CP-induced ototoxicity is increased levels of mitochondrial reactive oxygen species (ROS). In this study, we examined the effect of 2-Isopropyl-3H-naphtho(1,2-d)imidazole-4,5-dione (KL1333), a ß-lapachone derivative, on CP-induced ototoxicity using ex vivo organotypic culture system of cochlea. Hair cell damages in CP-treated cochlear explants with or without KL1333 were compared by immunohistochemistry. CP-induced oxidative stress and the preventive effect of KL1333 were analyzed by measuring intracellular ROS levels and depolarization of mitochondrial membrane potential. Activation of apoptosis signaling pathway was detected using TUNEL assay and immunostaining of cleaved caspase-3. As the results, it was found that KL1333 pretreatment significantly decreased stereocilia degeneration and hair cell loss, and prevented an increase in mitochondrial ROS levels in response to CP. Immunohistochemical examinations of cochlear explants revealed greater caspase-3 immunopositivity in the CP group than in controls, while the KL1333 + CP group showed significantly less immunopositivity than the CP group (P < 0.05). Thus, it appeared that KL1333 protected hair cells in the organ of Corti from CP-induced apoptosis by decreasing mitochondrial damages due to the production of mitochondrial ROS. This study is the first report showed the preventive effect of KL1333 against CP-induced ototoxicity. Although further studies should be performed to determine if KL1333 could maintain anticancer effect of CP, our data cautiously suggests that the antioxidant KL1333 can be used as an effective anti-apoptotic agent to prevent ototoxicity caused by CP-induced oxidative stress, and may prove useful in preventing hearing loss caused by CP.

Modified U1 snRNA and antisense oligonucleotides rescue splice mutations in SLC26A4 that cause hereditary hearing loss.

One of most important factors for messenger RNA (mRNA) transcription is the spliceosomal component U1 small nuclear RNA (snRNA), which recognizes 5' splicing donor sites at specific regions in pre-mRNA. Mutations in these sites disrupt U1 snRNA binding and cause abnormal splicing. In this study, we investigated mutations at splice sites in SLC26A4 (HGNC 8818), one of the major causative genes of hearing loss, which may result in the synthesis of abnormal pendrin, the channel protein encoded by the gene. Seventeen SLC26A4 variants with mutations in the U1 snRNA binding sites were assessed by minigene splicing assays, and 11 were found to result in abnormal splicing. Interestingly, eight of the 11 pathogenic mutations were intronic, suggesting the importance of conserved sequences at the intronic splice site. The application of modified U1 snRNA effectively rescued the abnormal splicing for most of these mutations. Although three were cryptic mutations, they were rescued by cotransfection of modified U1 snRNA and modified antisense oligonucleotides. Our results demonstrate the important role of snRNA in SLC26A4 mutations, suggesting the therapeutic potential of modified U1 snRNA and antisense oligonucleotides for neutralizing the pathogenic effect of the splice-site mutations that may result in hearing loss.

Correction to: Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.

The original article contains an error for a grant number in the Acknowledgements section.

Therapeutic potential of the mitochondria-targeted antioxidant MitoQ in mitochondrial-ROS induced sensorineural hearing loss caused by Idh2 deficiency.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major NADPH-producing enzyme which is essential for maintaining the mitochondrial redox balance in cells. We sought to determine whether IDH2 deficiency induces mitochondrial dysfunction and modulates auditory function, and investigated the protective potential of an antioxidant agent against reactive oxygen species (ROS)-induced cochlear damage in Idh2 knockout (Idh2-/-) mice. Idh2 deficiency leads to damages to hair cells and spiral ganglion neurons (SGNs) in the cochlea and ultimately to apoptotic cell death and progressive sensorineural hearing loss in Idh2-/- mice. Loss of IDH2 activity led to decreased levels of NADPH and glutathione causing abnormal ROS accumulation and oxidative damage, which might trigger apoptosis signal in hair cells and SGNs in Idh2-/- mice. We performed ex vivo experiments to determine whether administration of mitochondria-targeted antioxidants might protect or induce recovery of cells from ROS-induced apoptosis in Idh2-deficient mouse cochlea. MitoQ almost completely neutralized the H2O2-induced ototoxicity, as the survival rate of Idh2-/- hair cells were restored to normal levels. In addition, the lack of IDH2 led to the accumulation of mitochondrial ROS and the depolarization of ΔΨm, resulting in hair cell loss. In the present study, we identified that IDH2 is indispensable for the functional maintenance and survival of hair cells and SGNs. Moreover, the hair cell degeneration caused by IDH2 deficiency can be prevented by MitoQ, which suggests that Idh2-/- mice could be a valuable animal model for evaluating the therapeutic effects of various antioxidant candidates to overcome ROS-induced hearing loss.

Evaluating protective and therapeutic effects of alpha-lipoic acid on cisplatin-induced ototoxicity.

Cisplatin, a small platinum-containing molecule, is a widely used, highly effective anticancer drug. However, severe side effects have been found in cancer patients treated with cisplatin, including nephrotoxicity, neurotoxicity, and ototoxicity. These cisplatin-induced side effects can have a major impact on patient quality of life, including social development problems in pediatric patients that develop hearing loss. Previous studies have suggested that the major cause of cisplatin-induced ototoxicity is abnormal accumulation of reactive oxygen species (ROS) and oxidative stress. Alpha-lipoic acid (ALA), one of the most effective antioxidants, is known to be involved in the cellular antioxidant system and may have a protective effect on cisplatin-induced ototoxicity. However, the therapeutic effect of ALA on damaged hearing function and its detailed mechanism of action are not fully understood. This study focused on determining whether ALA has a potential as a protective and/or therapeutic agent for cisplatin-induced ototoxicity. Histological and physiological analyses were performed using cisplatin-treated mouse cochlea and HEI-OC1 culture cells in pre- and post-treatment with ALA in vitro and in vivo. We found that ALA contributes to protecting mitochondrial function by preventing ROS accumulation and inhibiting apoptotic cell death. Importantly, post-treatment with ALA consistently showed an almost equal restorative effect to pretreatment, in vitro and in vivo, supporting the possible use of ALA as a therapeutic agent for cisplatin-induced ototoxicity. This study is the first report on a strong therapeutic potential of ALA to rescue ototoxic hearing loss caused by cisplatin, and our data provide key evidence that ALA may act as a reducing agent for glutathione disulfide to increase glutathione levels on behalf of glutathione reductase. This result was consistent in both cultured cells and the mouse model, which improves the clinical value of ALA for therapy of cisplatin-induced ototoxicity.

Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy.

The-state-of-art CRISPR/Cas9 is one of the most powerful among the approaches being developed to rescue fundamental causes of gene-based inheritable diseases. Several strategies for delivering such genome editing materials have been developed, but the safety, efficacy over time, cost of production, and gene size limitations are still under debate and must be addressed to further improve applications. In this study, we evaluated branched forms of the polyethylenimine (PEI) - branched PEI 25 kDa (BPEI-25K) - and found that it could efficiently deliver CRISPR/Cas9 plasmids. Plasmid DNA expressing both guide RNA and Cas9 to target the Slc26a4 locus was successfully delivered into Neuro2a cells and meditated genome editing within the targeted locus. Our results demonstrated that BPEI-25K is a promising non-viral vector to deliver the CRISPR/Cas9 system in vitro to mediate targeted gene therapy, and these findings contribute to an understanding of CRISPR/Cas9 delivery that may enable development of successful in vivo techniques.

Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1Cre mice with Exoc5f/f mice for hair cell-specific deletion (Gfi1Cre/+;Exoc5f/f) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5f/f). Gfi1Cre/+;Exoc5f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.

Identification of a novel splicing mutation within SLC17A8 in a Korean family with hearing loss by whole-exome sequencing.

Hereditary hearing loss (HHL) is a common genetically heterogeneous disorder, which follows Mendelian inheritance in humans. Because of this heterogeneity, the identification of the causative gene of HHL by linkage analysis or Sanger sequencing have shown economic and temporal limitations. With recent advances in next-generation sequencing (NGS) techniques, rapid identification of a causative gene via massively parallel sequencing is now possible. We recruited a Korean family with three generations exhibiting autosomal dominant inheritance of hearing loss (HL), and the clinical information about this family revealed that there are no other symptoms accompanied with HL. To identify a causative mutation of HL in this family, we performed whole-exome sequencing of 4 family members, 3 affected and an unaffected. As the result, A novel splicing mutation, c.763+1G>T, in the solute carrier family 17, member 8 (SLC17A8) gene was identified in the patients, and the genotypes of the mutation were co-segregated with the phenotype of HL. Additionally, this mutation was not detected in 100 Koreans with normal hearing. Via NGS, we detected a novel splicing mutation that might influence the hearing ability within the patients with autosomal dominant non-syndromic HL. Our data suggests that this technique is a powerful tool to discover causative genetic factors of HL and facilitate diagnoses of the primary cause of HHL.

A novel missense variant in the DIAPH1 gene in a Korean family with autosomal dominant nonsyndromic hearing loss.

Hair cells in the cochlea display highly regulated actin polymerization, which is mediated by the human diaphanous-related formin 1 gene (DIAPH1; also called DFNA1, DIA1). DFNA1, the first type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is known to be associated with mutations in DIAPH1. However, no genetic study of DFNA1 in Koreans with hearing loss has yet been reported. A 51-year-old patient in a Korean family with ADNSHL was examined by pure-tone audiometry, and genetic analysis of DIAPH1 was performed. A novel variant, p.I530S (c.1589T > G), was identified in the DIAPH1 gene, and the mutation was located in the highly conserved coiled-coil domain of the DIA1 protein, where an amino acid substitution was predicted to change the domain structure. Further functional investigations will provide more information to help us understand the role of DIAPH1 in maintenance of hair cell function in the auditory pathway.

A Novel Frameshift Mutation of SLC26A4 in a Korean Family With Nonsyndromic Hearing Loss and Enlarged Vestibular Aqueduct.

OBJECTIVES: We aimed to identify the causative mutation for siblings in a Korean family with nonsyndromic hearing loss (HL) and enlarged vestibular aqueduct (EVA). The siblings were a 19-year-old female with bilateral profound HL and an 11-year-old male with bilateral moderately severe HL. METHODS: We extracted genomic DNA from blood samples of the siblings with HL, their parents, and 100 controls. We performed mutation analysis for SLC26A4 using direct sequencing. RESULTS: The two siblings were compound heterozygotes with the novel mutation p.I713LfsX8 and the previously described mutation p.H723R. Their parents had heterozygous mono-allelic mutations. Father had p.I713LfsX8 mutation as heterozygous, and mother had p.H723R mutation as heterozygous. However, novel mutation p.I713LfsX8 was not detected in 100 unrelated controls. CONCLUSION: Both mutations identified in this study were located in the sulfate transporter and anti-sigma factor antagonist domain, the core region for membrane targeting of SulP/SLC26 anion transporters, which strongly suggests that failure in membrane trafficking by SLC26A4 is a direct cause of HL in this family. Our study could therefore provide a foundation for further investigations elucidating the SLC26A4-related mechanisms of HL.

Genetic association of MYH genes with hereditary hearing loss in Korea.

BACKGROUND: Myosin is a key protein involved in regulating the shape and motility of cells. The MYH9 and MYH14 genes, which encode non-muscle myosin heavy chain IIA (NMMHC II-A) and IIC (NMMHC II-C), respectively, are expressed in the inner ear. These myosin genes are known to be associated with autosomal dominant non-syndromic hearing loss (ADNSHL); however, genetic studies in patients with ADNSHL in Korea have rarely been reported. METHODS: We analyzed the MYH9 and MYH14 genes in 75 Korean patients with ADNSHL. RESULTS: We identified 4 possible pathogenic variants: a novel variant p.F1303L and 2 previously reported variants (p.R1730C and p.R1785C) in the MYH9 gene, and a novel variant p.A1868T in the MYH14 gene. All the variants were located in the myosin tail domain, which is essential for the interaction of myosin with actin. These variants were predicted to be possibly pathogenic by functional prediction tools and were absent in 100 unrelated normal controls. CONCLUSION: These results suggest that all the variants identified in this study have a strong potential to affect the structural stability and/or function of non-muscle myosin in the inner ear, which might lead to ADNSHL. This study establishes the link between the genotype and development of ADNSHL and contributes to the establishment of Korean database for hereditary hearing loss.

Dynamin Binding Protein (Tuba) Deficiency Inhibits Ciliogenesis and Nephrogenesis in Vitro and in Vivo.

Dysfunction of renal primary cilia leads to polycystic kidney disease. We previously showed that the exocyst, a protein trafficking complex, is essential for ciliogenesis and regulated by multiple Rho and Rab family GTPases, such as Cdc42. Cdc42 deficiency resulted in a disruption of renal ciliogenesis and a polycystic kidney disease phenotype in zebrafish and mice. Here we investigate the role of Dynamin binding protein (also known as Tuba), a Cdc42-specific guanine nucleotide exchange factor, in ciliogenesis and nephrogenesis using Tuba knockdown Madin-Darby canine kidney cells and tuba knockdown in zebrafish. Tuba depletion resulted in an absence of cilia, with impaired apical polarization and inhibition of hepatocyte growth factor-induced tubulogenesis in Tuba knockdown Madin-Darby canine kidney cell cysts cultured in a collagen gel. In zebrafish, tuba was expressed in multiple ciliated organs, and, accordingly, tuba start and splice site morphants showed various ciliary mutant phenotypes in these organs. Co-injection of tuba and cdc42 morpholinos at low doses, which alone had no effect, resulted in genetic synergy and led to abnormal kidney development with highly disorganized pronephric duct cilia. Morpholinos targeting two other guanine nucleotide exchange factors not known to be in the Cdc42/ciliogenesis pathway and a scrambled control morpholino showed no phenotypic effect. Given the molecular nature of Cdc42 and Tuba, our data strongly suggest that tuba and cdc42 act in the same ciliogenesis pathway. Our study demonstrates that Tuba deficiency causes an abnormal renal ciliary and morphogenetic phenotype. Tuba most likely plays a critical role in ciliogenesis and nephrogenesis by regulating Cdc42 activity.

Identification of a nonsense mutation in the STRC gene in a Korean family with moderate hearing loss.

Hereditary hearing loss is a heterogeneous disorder that results in a common sensorineural disorder. To date, more than 150 loci and 89 genes have been reported for non-syndromic hearing loss. Next generation sequencing has recently been developed as a powerful genetic strategy for identifying pathogenic mutations in heterogeneous disorders with various causative genes. In this study, we performed targeted sequencing to identify the causative mutation in a Korean family that had moderate hearing loss. We targeted 64 genes associated with non-syndromic hearing loss and sorted the homozygous variations according to the autosomal recessive inheritance pattern of the family. Implementing a bioinformatic platform for filtering and detecting variations allowed for the identification of two variations within different genes (c.650G>A in TRIOBP and c.4057C>T in STRC). These variants were selected for further analysis. Among these, c.4057C>T (p.Q1353X) was a divergent sequence variation between the STRC gene and the STRC pseudogene. This was the critical difference that resulted in loss of the protein-coding ability of the pseudogene. Therefore, we hypothesized that the p.Q1353X variation in the STRC gene is the causative mutation for hearing loss. This result suggests that application of targeted sequencing will be valuable for the diagnosis of heterogeneous disorders.

Pannexin 3 is required for normal progression of skeletal development in vertebrates.

The vertebrate skeletal system has various functions, including support, movement, protection, and the production of blood cells. The development of cartilage and bones, the core components of the skeletal system, is mediated by systematic inter- and intracellular communication among multiple signaling pathways in differentiating progenitors and the surrounding tissues. Recently, Pannexin (Panx) 3 has been shown to play important roles in bone development in vitro by mediating multiple signaling pathways, although its roles in vivo have not been explored. In this study, we generated and analyzed Panx3 knockout mice and examined the skeletal phenotypes of panx3 morphant zebrafish. Panx3(-/-) embryos exhibited delays in hypertrophic chondrocyte differentiation and osteoblast differentiation as well as the initiation of mineralization, resulting in shortened long bones in adulthood. The abnormal progression of hypertrophic chondrogenesis appeared to be associated with the sustained proliferation of chondrocytes, which resulted from increased intracellular cAMP levels. Similarly, osteoblast differentiation and mineralization were delayed in panx3 morphant zebrafish. Taken together, our results provide evidence of the crucial roles of Panx3 in vertebrate skeletal development in vivo.

A post-developmental genetic screen for zebrafish models of inherited liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease.