The deafness gene dfna5 is crucial for ugdh expression and HA production in the developing ear in zebrafish.
ABSTRACT Over 30 genes responsible for human hereditary hearing loss have been identified during the last 10 years. The proteins encoded by these genes play roles in a diverse set of cellular functions ranging from transcriptional regulation to K(+) recycling. In a few cases, the genes are novel and do not give much insight into the cellular or molecular cause for the hearing loss. Among these poorly understood deafness genes is DFNA5. How the truncation of the encoded protein DFNA5 leads to an autosomal dominant form of hearing loss is not clear. In order to understand the biological role of Dfna5, we took a reversegenetic approach in zebrafish. Here we show that morpholino antisense nucleotide knock-down of dfna5 function in zebrafish leads to disorganization of the developing semicircular canals and reduction of pharyngeal cartilage. This phenotype closely resembles previously isolated zebrafish craniofacial mutants including the mutant jekyll. jekyll encodes Ugdh [uridine 5'-diphosphate (UDP)-glucose dehydrogenase], an enzyme that is crucial for production of the extracellular matrix component hyaluronic acid (HA). In dfna5 morphants, expression of ugdh is absent in the developing ear and pharyngeal arches, and HA levels are strongly reduced in the outgrowing protrusions of the developing semicircular canals. Previous studies suggest that HA is essential for differentiating cartilage and directed outgrowth of the epithelial protrusions in the developing ear. We hypothesize that the reduction of HA production leads to uncoordinated outgrowth of the canal columns and impaired facial cartilage differentiation.
- SourceAvailable from: Joe Sisneros[Show abstract] [Hide abstract]
ABSTRACT: Zebrafish (Danio rerio) have become a valuable model for investigating the molecular genetics and development of the inner ear in vertebrates. In this study, we employed a prepulse inhibition (PPI) paradigm to assess hearing in larval wild-type (AB) zebrafish during early development at 5-6 days post-fertilization (d.p.f.). We measured the PPI of the acoustic startle response in zebrafish using a 1-dimensional shaker that simulated the particle motion component of sound along the fish's dorsoventral axis. The thresholds to startle-inducing stimuli were determined in 5-6 d.p.f. zebrafish, and their hearing sensitivity was then characterized using the thresholds of prepulse tone stimuli (90-1200 Hz) that inhibited the acoustic startle response to a reliable startle stimulus (820 Hz at 20 dB re. 1 m s(-2)). Hearing thresholds were defined as the minimum prepulse tone level required to significantly reduce the startle response probability compared with the baseline (no-prepulse) condition. Larval zebrafish showed greatest auditory sensitivity from 90 to 310 Hz with corresponding mean thresholds of -19 to -10 dB re. 1 m s(-2), respectively. Hearing thresholds of prepulse tones were considerably lower than previously predicted by startle response assays. The PPI assay was also used to investigate the relative contribution of the lateral line to the detection of acoustic stimuli. After aminoglycoside-induced neuromast hair-cell ablation, we found no difference in PPI thresholds between treated and control fish. We propose that this PPI assay can be used to screen for novel zebrafish hearing mutants and to investigate the ontogeny of hearing in zebrafish and other fishes.Journal of Experimental Biology 09/2013; 216(Pt 18):3504-13. DOI:10.1242/jeb.087635 · 3.00 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Gasdermin-like (GSDML) belongs to the gasdermin-domain-containing protein family (GSDMDC family) that is involved in carcinogenesis and hearing impairment. However, the role of GSDML in carcinogenesis remains unclear. In this study, we identified four isoforms of GSDML gene. The primary and longest isoform GSDML1 is widely expressed in human cancer cell lines. GFP-GSDML1 fusion protein was localized predominantly in the nucleus of human breast cancer MCF7 and cervical cancer HeLa cells but exclusively in the cytoplasm of hepatocellular carcinoma HepG2 cells. Importantly, immunohistochemistry analysis showed that the GSDML protein in the nuclei is expressed at a higher level in uterine cervix cancer tissues than in the adjacent cancer tissues and corresponding nonneoplastic tissues. Such significance was not observed in hepatocellular carcinoma tissues. Ectopic expression of GSDML1 enhanced the growth of cultured cells, whereas inhibition of its endogenous expression decreased proliferation. Furthermore, GSDML1 had significant effects on promoting bromodeoxyuridine incorporation in cells. However, GSDML1 could neither promote malignant transformation nor gain the ability of colony formation or carcinogenicity on nude mice. Collectively, these results suggest that GSDML can promote cell proliferation, and it might be correlated with carcinogenesis and progression of uterine cervix cancer.Translational oncology 07/2008; 1(2):73-83. DOI:10.1593/tlo.08112 · 3.40 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.Developmental Biology 07/2006; 294(1):148-60. DOI:10.1016/j.ydbio.2006.02.034 · 3.64 Impact Factor