Ethanol affects the development of sensory hair cells in larval zebrafish (Danio rerio) - PubMed (original) (raw)

Ethanol affects the development of sensory hair cells in larval zebrafish (Danio rerio)

Phillip M Uribe et al. PLoS One. 2013.

Abstract

Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS). The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf) and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%-1.75% by volume) from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.

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Conflict of interest statement

Competing Interests: The senior author of this manuscript, Jonathan I. Matsui, is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials as outlined in the guide for authors.

Figures

Figure 1

Figure 1. Ethanol treatment affects the development of larval zebrafish.

Zebrafish embryos were raised in varying concentrations of ethanol (0% through 1.5% by volume) from 2 days post-fertilization (dpf) to 5 dpf, fixed, and imaged. (A) Untreated control animals appear normal. (B) 1% ethanol-treated animals have a slightly swollen heart (arrow) and no swim bladder but otherwise appear normal. (C) Increasing the concentration of ethanol treatment to 1.5% resulted in embryos exhibiting swollen hearts (arrow), swollen guts (#), missing a swim bladder (white asterisk), and inhibited craniofacial development (arrowhead), which is similar to the phenotype exhibited by children with FAS.

Figure 2

Figure 2. Ethanol but not methanol treatment affects the number of functional sensory hair cells in the lateral line.

Brn3c-GFP transgenic zebrafish embryos were raised in embryo medium supplemented with varying concentrations of ethanol. Prior to fixation, the larvae were stained with FM 1-43FX (red) to determine if hair cell mechanotransduction channels were functional. (A) Lateral view of a Brn3c-GFP zebrafish. Clusters of bright green GFP-labeled hair cells are found in neuromasts along the head and body of the animal. The O2 and Mi1 neuromasts are highlighted along with inner ear organs (small arrows). Higher magnification of hair cells of the O2 neuromast taken from _z_-stacks show that (B) hair cells in untreated controls and (C) 1% ethanol-treated animals had no observable morphological differences though there were fewer hair cells in the 1% ethanol-treated animals. Almost all of the GFP-labeled hair cells were co-stained with FM 1-43FX. (D) Fewer sensory hair cells were observed in neuromasts of animals treated with 1.5% ethanol. There were also fewer double-labeled cells (arrowhead) and more single labeled cells (asterisk). (E) Stereocilia bundles were counted to determine the number of hair cells present in the O2 neuromast. Control numbers were similar to those reported in other studies , . Significantly fewer hair cells (**p<0.01) were counted in ethanol-treated larvae at concentrations greater than 1% per volume when compared to controls. No differences in the number of hair cells were observed in larvae treated with 1.5% methanol. (F) The percentage of GFP-labeled cells that were also co-stained for FM 1-43 decreased as the concentration of ethanol increased but not in methanol-treated larvae. There was a significant decrease (**p<0.01) in the number of double-labeled hair cells at the two highest concentrations of ethanol tested. Results are the mean values ± SD. n = 8-21 per condition.

Figure 3

Figure 3. Ethanol exposure significantly reduced the number of proliferating cells compared to untreated controls in O2 neuromasts of Brn3c GFP-larvae.

(A) Under untreated control conditions, proliferating cell nuclear antigen (PCNA)-labeled cells (arrowhead) are primarily non-sensory supporting cells and mantle cells in the neuromast (area demarked by white dotted line). Fewer PCNA-labeled cells result from ethanol treatment at (B) 1% and (C) 1.5% by volume. (D) The mean number of PCNA-labeled cells decreases during maturation and according dose of ethanol. (E) In a separate experiment, bromodeoxyuridine was added to the embryo medium during the last 24 hours of treatment, fixed, processed for BrdU immunohistochemistry, and BrdU-labeled cells were counted. Significantly fewer BrdU-labeled cells were observed in ethanol-treated animals when compared to untreated controls. Results are the mean values ± SD. n = 10-21 neuromasts for each treatment group. **p<0.01.

Figure 4

Figure 4. Ethanol exposure significantly increased the number of TUNEL-labeled cells in the O2 neuromast.

Apoptotic activity in O2 neuromasts was measured by TUNEL labeling of untreated and ethanol-treated Brn3c-GFP larvae from 2-5 dpf. (A, D) Untreated controls showed few TUNEL-labeled cells but (B, D) 1% ethanol-treated and (C, D) 1.5% ethanol-treated larvae showed a significant increase in the number of TUNEL-labeled cells (arrowhead) versus control animals. While there were more TUNEL-labeled cells in ethanol-treated animals when compared to untreated controls, there was no statistical difference between ethanol concentrations. Results are the mean values ± SD. n = 9-12 neuromasts for each treatment group. *p<0.05 and **p<0.01 when compared to untreated controls.

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