Genetic Analysis of Vertebrate Sensory Hair Cell Mechanosensation: the Zebrafish Circler Mutants

Abstract

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

Early Development of Hearing in Zebrafish

“…After the microelectrode tip penetrated into the otic vesicle, the FM1-43X solution was pressure injected into the otic vesicle with a 10-ml syringe connected to the pressure port of the electrode holder. The injection was monitored under the Zeiss Axioskop microscope with the 40× water immersion objective lens and a Texas Red filter set in order to verify hair cell uptake of the dye, which is known to selectively and rapidly label sensory hair cells and then block their responses, presumably by entering mechanotransduction ion channels (Nishikawa and Sasaki 1996;Nicolson et al 1998;Gale et al 2001;Meyers et al 2003). FM1-43X was used as a control to rule out stimulus artifact as shown in Fig.…”

“…Compared with mammals (see a review by Rübsamen and Lippe 1998), the zebrafish is a unique model for the study of early development of hearing and balance in vertebrates because the inner ear of zebrafish embryos is easily accessible for live imaging, physiological recording, and genetic manipulation (Waterman and Bell 1984;Haddon and Lewis 1996;Nicolson et al 1998;Bever and Fekete 2002;Whitfield 2002;Nicolson 2005;Zeddies and Fay 2005;Tanimoto et al 2011). Previous studies have reported on the development of auditory sensitivity in a few juvenile and adult teleosts using various physiological methods (Corwin 1983;Kenyon 1996;Higgs et al 2003;Sisneros and Bass 2005;Vasconcelos and Ladich 2008;Alderks and Sisneros 2011;Lechner et al 2012).…”

“…Finally, the zebrafish genome has been sequenced, and many human genes are conserved in zebrafish. Particularly, the zebrafish has been used as a unique vertebrate model for studying hearing and balance disorders (Nicolson et al 1998;Ernest et al 2000;Whitfield 2002;Söllner et al 2004;Kappler et al 2004;Nicolson 2005;Shen et al 2008;Gleason et al 2009;Phillips et al 2011;Yariz et al 2012), with its externally exposed hair cells of the lateral line being used for study in hair cell death and regeneration, ototoxicity, and drug screens (Chiu et al 2008;López-Schier et al 2004;Owens et al 2009;Coffin et al 2010;Buck et al 2012).…”

“…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.…”

Behavioral genetic approaches to visual system development and function in zebrafish

“…This simple behavior has been used to measure the spectral sensitivity and the visual sensitivity of this behavior in the adult goldfish visual system (Powers, 1978;Yager, 1968). In zebrafish, the DLR is only obvious in mutants with defects in their sense of balance (Nicolson et al, 1998). The response becomes much more pronounced when the fish is placed head down in a tightly fitting water-filled tube.…”