Rheotaxis in Larval Zebrafish Is Mediated by Lateral Line Mechanosensory Hair Cells

Citation: Suli A, Watson GM, Rubel EW, Raible DW ( Rheotaxis in Larval Zebrafish Is Mediated by Lateral Line Mechanosensory Hair Cells Arminda Suli 0 Glen M. Watson 0 Edwin W. Rubel 0 David W. Raible 0 Bruce Riley, Texas A&M University, United States of America 0 1 Department of Biological Structure, University of Washington, Seattle, Washington, United States of America, 2 Department of Biology, University of Louisiana Lafayette, Lafayette, Louisiana, United States of America, 3 V. M. Bloedel Hearing Research Center, University of Washington , Seattle, Washington , United States of America The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration. - Funding: This work was supported by R01DC005987 to DWR from the National Institute on Deafness and Other Communication Disorders (NIDCD) (www.nidcd. nih.gov), and a research grant from the Hearing Health Foundation (www.drf.org) to AS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Mechanosensory hair cells are specialized cells with protruding apical stereocilia whose deflection leads to voltage changes that are transmitted to the central nervous system. The auditory system, vestibular system and mechanosensory lateral line system utilize these sensory cells as the first stage of processing information about the environment. The lateral line system, present in fish and amphibians, allows these organisms to respond to mechanical stimuli generated from water motion in their surroundings. This system is composed of sensory organs named neuromasts, which are located in canals or deposited on the body surface of the developing larvae [1,2]. The mechanosensory hair cells reside in the center of neuromasts and are surrounded by interdigitating glial-like support cells [2,3]. Behaviors such as schooling [4], prey detection [5,6] and escape [7,8] have been shown to be dependent on the proper function of the lateral line system. Unlike their mammalian counterparts, hair cells of birds [9,10], amphibians [3,11] and fish [12,13,14,15] regenerate following cell damage [16]. The easily accessible mechanosensory lateral line has emerged as an excellent system in which to study hair cell death and regeneration in the hopes of understanding the mechanisms underlying these processes and identifying potential therapeutic solutions for hearing and balance disorders. Since lateral line mechanosensory hair cells are able to regenerate, developing a functional assay following hair cell perturbation is of particular interest. In adult fish, rheotaxis, the ability to align against current, partially depends on the lateral line system [17,18,19]. While there is evidence that larval zebrafish can swim under flow conditions [20,21], it is not known to what extent larval rheotaxis depends on lateral line function. To address this issue, we developed methods to evaluate rheotaxis under constant flow conditions. We then varied flow rate, lighting conditions and presence, damage, absence or regeneration of lateral line hair cells and tested their effect on rheotaxis. Of most interest, we report that of hair cells or damage to stereocilia bundle integrity disrupts rheotaxis, and that normal rheotaxis resumes upon repair of bundle integrity or with regeneration of hair cells. Rheotaxis apparatus and assay A 1.1 m length63.7 cm width65.1 cm height clear plastic flume was connected to a peristaltic pump (Dynamax, RP-1, Rainin,) to create a closed flow system (Figure 1A). The flume was filled with E2 (embryo medium) [22] to a depth of 2.8 cm, and the pump generated flow up to 0.2 cm/s. Two stainless steel screens were placed 5 cm apart toward the middle of the flume, partitioning an observation area. We initially placed drinking straws upstream of the observation chamber to act as collimators, but found that the screens were sufficient to create laminar flow within the observation area. To test for laminar flow, we applied Figure 1. Zebrafish larvae can rheotax in flow conditions. A) We built a clear plastic, rectangular flume in which to test rheotaxis. 2030 larvae are placed in observation chamber and their alignment is recorded. B) Using ImageJ (NIH) macros, images are cropped, ellipses are fitted around each larva, and their alignment angle against current is calculated. Larvae display a preference for aligning against current in flow conditions (0.14 cm/s) as shown by frequency distribution (D), mean (E) and median (F) of alignment. C) Larvae show no left-right preference; therefore, their alignment is calculated in a 0180u scale (C). Experiment performed in IR. doi:10.1371/journal.pone.0029727.g001 2% phenol red upstream of the first screen and imaged dye movement from the side. We observed a boundary layer effect in the bottom 0.5 cm of the flume that caused no turbulence to the rest of the column. A Dage-MTI (Michigan City, IN) CCD72 camera was placed above the observation chamber for imaging. A water heater (Visi-therm 25 W, Aquarium Systems, Mentor, OH) was placed downstream of the observation chamber to maintain temperature of re-circulating medium between 2628uC. In experiments in which we sought to eliminate visual input to larval, we captured images under infrared illumination, lighting the chamber from above with an IR LED light array (920 nm IRTILE, PolarisUSA, Norcross, GA). We used the AB strain in all experiments. Embryos were collected and kept at 28uC in a 14 hr/10 hr light/dark cycle. Larvae were assayed at 5 days post fertilization (dpf), when they reached ,0.33 cm in length. 5 dpf larvae were dark-adapted for at least 30 min (EDTA experiments) or 34 hr (all other experiments) prior to testing. All experiments were performed at the same time of day since larval locomotor activity is affected by circadian rhyth (...truncated)