Mutation of the zebrafish choroideremia gene encoding Rab escort protein 1 devastates hair cells

Abstract

To identify genes important for hair-cell function, we conducted a mutagenic screen in zebrafish. Larvae from one mutant line, ru848, were unresponsive to acoustic stimuli and unable to balance. The mutation results in a 90% reduction in hair-cell number and partial retinal degeneration by 5 days postfertilization. We localized the recessive ru848 mutation by positional cloning to the zebrafish homolog of the human Choroideremia gene, which encodes Rab escort protein 1. This protein is essential for the normal prenylation of Rabs. Mutations in the human gene induce choroideremia, a disease marked by slow-onset degeneration of rod photoreceptors and retinal pigment epithelial cells. The degenerative phenotype resulting from a null mutation in the zebrafish gene indicates that hair cells and retinal cells require Rab escort protein 1 for survival. The internal ear, one of the most complex organs of a vertebrate organism, converts mechanical stimuli into neuronal responses. The task of mechanotransduction is accomplished by the ear's sensory receptor, the hair cell. The lateralline organs of fishes and aquatic amphibians also use this receptor to detect water movements. Despite our detailed knowledge of the hair cell's mechanical and electrical properties (1), our progress in understanding its development and operation has been impeded by our lack of knowledge of the relevant molecular components.One strategy used to identify genes important for hair-cell function is the investigation of their loss-of-function phenotypes, deafness and disequilibrium. The principal advantage of such a genetic approach is that detailed characterization of the mutant phenotype provides explicit clues about the role of the gene involved. Large-scale mutagenesis screens for mechanosensory mutants in Drosophila melanogaster and Caenorhabditis elegans have identified molecules critical for vibration and touch sensitivity (2-4). The absence of hair cells in invertebrates, however, makes screens specific to hair-cell defects impossible in these organisms.To identify important proteins in the hair cell, we have created point mutations randomly throughout the zebrafish genome and screened for impairment of ear and lateral-line-organ development and function. Several of the identified mutations have been subjected to further phenotypic analysis and genetic mapping and cloning. We report here the characterization of a mutation that identifies an important process in hair cells and serves as a model of degenerative disease in the human retina. Materials and Methods ru848Identification and Breeding. Zebrafish were maintained in aquaria (Marine Biotech, Beverly, MA) according to published protocols (5). Embryos were raised in a 28.5°C incubator in facility water containing 1 g͞ml methylene blue and staged as described (6). Male fish of the TL strain were mutagenized with ethylnitrosourea and bred for three generations to create homozygous F 3 larvae (7) that were screened for hearing defects by observing their response to a sharp tap on the side o...

Early Development of Hearing in Zebrafish

“…The displacement of the stimulus probe was always set at 5.8 μm during the experiments unless otherwise specified in the text. The probe tip was placed against the skin at the posterior end of the otic vesicle (pointing to the saccular otolith) and provided linear oscillatory motion along an axis 20 o off the longitudinal axis of the fish body (Corey et al 2004;Starr et al 2004). The parameters such as size, angle, and location of the stimulus probe affecting microphonic responses were kept constant for all recordings reported herein.…”

“…Particularly, Fay's shaker apparatus was used to accelerate the whole fish, resulting in acceleration of the inner ear, to simulate underwater acoustic particle motion (Fay 1984;. In this study, we adapted a stimulus probe from Corey et al (2004) and Starr et al (2004) to provide focal stimulation directly to the inner ear of zebrafish embryos or larvae instead of accelerating the entire fish body. The oscillatory motion of the probe tip caused the vibration of the posterior wall of the otic vesicle, leading to auditory transduction of hair cells in the inner ear.…”

“…Therefore, zebrafish during the first week of development are not likely sensitive to the pressure mode of sound because they lack the Weberian ossicles and/or the swim bladder to convey pressure stimulation to the inner ear. In this study, a stimulus probe was developed to directly stimulate the inner ear of zebrafish embryos or larvae (Corey et al 2004;Starr et al 2004), which simulates acoustic particle motion of sound. The microphonic potential method was successfully applied to young wild-type and mutant/ morphant zebrafish (Nicolson et al 1998;Starr et al 2004;Corey et al 2004;Tanimoto et al 2009;Trapani and Nicolson 2010), making it a quick and useful tool to assess hair cell function in the zebrafish model.…”

“…In this study, a stimulus probe was developed to directly stimulate the inner ear of zebrafish embryos or larvae (Corey et al 2004;Starr et al 2004), which simulates acoustic particle motion of sound. The microphonic potential method was successfully applied to young wild-type and mutant/ morphant zebrafish (Nicolson et al 1998;Starr et al 2004;Corey et al 2004;Tanimoto et al 2009;Trapani and Nicolson 2010), making it a quick and useful tool to assess hair cell function in the zebrafish model. In this study, we report the development of auditory function of zebrafish during the first week post fertilization by recording microphonic potentials from the inner ear and reveal the structural basis underlying the development of auditory function.…”

Zebrafish: A model system for the study of eye genetics

“…Similarly, two genes essential for the function of photoreceptor cells and associated with retinal dystrophy have recently been identified. The mutation in the zebrafish orthologue of the human choroideremia gene, which encodes the Rab escort protein-1 and is responsible for a human disease marked by slow-onset degeneration of rod photoreceptors and retinal pigment epithelial cells, was recently identified (Starr et al, 2004). Subsequent analysis suggested that the gene function is necessary in the RPE, and the photoreceptor deficit in secondary to this alteration (Krock, et al, 2007).…”