Modeling Environmentally-Induced Motor Neuron Degeneration in Zebrafish

Abstract

Zebrafish have been used to investigate motor neuron degeneration, including as a model system to examine the pathogenesis of amyotrophic lateral sclerosis (ALS). The use of zebrafish for this purpose has some advantages over other in vivo model systems. In the current paper, we show that bisphenol A (BPA) exposure in zebrafish embryos results in motor neuron degeneration with affected motor function, reduced motor axon length and branching, reduced neuromuscular junction integrity, motor neuron cell death and the presence of activated microglia. In zebrafish, motor axon length is the conventional method for estimating motor neuron degeneration, yet this measurement has not been confirmed as a valid surrogate marker. We also show that reduced motor axon length as measured from the sagittal plane is correlated with increased motor neuron cell death. Our preliminary timeline studies suggest that axonopathy precedes motor cell death. This outcome may have implications for early phase treatments of motor neuron degeneration.

Figure 1

Dose-dependent effect of BPA on motor axon length and branching on wild type embryos. (a) Motor axon length under increasing concentrations of BPA. (b) Total number of motor axon branching outgrowths under increasing concentrations of BPA. *P = 0.034, ***P < 0.001). Embryos were subject to 42 hours duration of exposure to different concentrations of BPA. Data are based on the mean value of N = 5–6 technical replicates (motor axon length), N = 2 technical replicates (motor axon branching) and N = 8–10 biological replicates. P values were determined by Kruskal-Wallis ranked sums and Mann-Whitney U post test.

Figure 2

BPA exposure impairs NMJ integrity. (a) Representative images of α-bungarotoxin (post-synaptic terminal) and synpatotagmin 2 (pre-synaptic terminal) labeling in wild type embryos at 48 hpf exposed to BPA vehicle control, or control media. Inset magnifications are merged images of the pre- and post-synaptic terminals. SC, spinal cord. (b) Data are based on the integrity of the NMJ at 48 hpf in control, vehicle control and BPA exposed embryos as measured by Pearson’s R² co-localization coefficient between α-bungarotoxin and synaptotagmin 2. P = 0.02, N = 8–15 biological replicates; P = 0.006, N = 15 biological replicates; N = 5–6 technical replicates of motor neurons. P values were determined by Mann-Whitney U test.

Figure 3

Activated microglia associate with defective motor neurons. Double transgenic Tg:mnx1-GFP/pU1-RFP embryos identify microglia (red) and motor neurons (green). White arrows point to pU1+ microglial cells spatially associated with motor neurons at 48 hpf. Percent values in the pie charts show the relative proportion of total microglia in each activation state compared to total number of microglia cells in region of interest (right panel) when exposed to BPA compared to vehicle controls (N = 9–12 biological replicates; N = 1–10 technical replicates). SC = spinal cord.

Figure 4

Motor neuron trajectory from a sagittal and cross sectional view. At 18 hpf, primary motor neurons start to exit the ventral root of the spinal cord. By 48 hpf each somite has one set of caudal (CaP), middle (MiP) and rostral (RoP) primary motor neurons innervating each side of the spinal segment. For simplicity, here we only show the CaP motor neuron. (a) CaP motor axons project ventrally from the SC to the ventral myotome. Axon length is conventionally measured from the sagittal plane, with the assumption that motor axons in control embryos and embryos with motor axon abnormalities follow the same trajectory. (b) Defective motor axons have a normal trajectory from the spinal cord. Tg:mnx1-GFP embryos exposed to BPA have a similar motor axonal trajectory through the Z axis (green) as vehicle controls (N = 3 biological replicates).

Figure 5

BPA exposure causes increased motor cell death at 72 hpf. (a) Motor neuron cell death is increased in the spinal cord starting at 72 hpf following BPA exposure as compared to vehicle controls. Values indicate colocalization coefficients between cells with a positive signal for both mnx1-GFP and PI in the spinal cord, indicating dead motor neurons. Group values between time points should be interpreted independently due to non-uniform PI staining between time points. (b) BPA-exposed embryos show colocalization between PI + cells and mnx1 motor neuron soma 96 hpf, and show that PI staining is not specific to mnx1 motor neuron soma. Data represent the mean ± s.d.; N = 7–12 biological replicates. P values were determined by Mann-Whitney U test.