XB-ART-50116Neurotoxicol Teratol January 1, 2015; 47 102-13.
Methylmercury exposure during early Xenopus laevis development affects cell proliferation and death but not neural progenitor specification.
Methylmercury (MeHg) is a widespread environmental toxin that preferentially and adversely affects developing organisms. To investigate the impact of MeHg toxicity on the formation of the vertebrate nervous system at physiologically relevant concentrations, we designed a graded phenotype scale for evaluating Xenopus laevis embryos exposed to MeHg in solution. Embryos displayed a range of abnormalities in response to MeHg, particularly in brain development, which is influenced by both MeHg concentration and the number of embryos per ml of exposure solution. A TC50 of ~50μg/l and LC50 of ~100μg/l were found when maintaining embryos at a density of one per ml, and both increased with increasing embryo density. In situ hybridization and microarray analysis showed no significant change in expression of early neural patterning genes including sox2, en2, or delta; however a noticeable decrease was observed in the terminal neural differentiation genes GAD and xGAT, but not xVGlut. PCNA, a marker for proliferating cells, was negatively correlated with MeHg dose, with a significant reduction in cell number in the forebrain and spinal cord of exposed embryos by tadpole stages. Conversely, the number of apoptotic cells in neural regions detected by a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was significantly increased. These results provide evidence that disruption of embryonic neural development by MeHg may not be directly due to a loss of neural progenitor specification and gene transcription, but to a more general decrease in cell proliferation and increase in cell death throughout the developing nervous system.
PubMed ID: 25496965
PMC ID: PMC4302163
Article link: Neurotoxicol Teratol
Species referenced: Xenopus laevis
Genes referenced: dll1 en2 gad1.2 gnl3 pcna slc17a7 sox2
Article Images: [+] show captions
|Fig. 1. Phenotype grading scale used for the visual analysis of the gross response of embryos to MeHg exposure. The scale applies to pre and post hatching embryos. The scores are: 5, normal development; 4, cell loss in vitelline membrane obscuring b 25% of the embryo without gross deformity for pre-, or minor deformities with no loss of morphology, such as spine curling for post-; 3, two or more stages behind paired non-exposed controls; 2, large amount of cell loss to vitelline membrane obscuring N25% of embryo for pre-, or large amount of deformity causing loss of morphological features in post-; 1, ball of cells that has ceased to develop. Not shown is score 0, which is full lysis and removal. Scale bar represents 1 mm.|
|Fig. 2. Stacked percentages of embryos in each post hatching phenotype score at stage 33 + for a given exposure level of MeHgCl in solution. (A) Percentage of scores for embryos exposed at a density of 1 e/ml. N = 12 experiments, with 134 embryos total on average per dose. (B) and (C) are percentages of scores for embryo densities of 2 e/ml and 3 e/ml respectively. N = 17 experiments, with 320 embryos total on average; and N = 6 experiments, with 172 embryos total on average per dose, respectively.|
|Fig. 3. Average response and MeHg uptake of embryos exposed to MeHgCl at a density of 1 e/ml. (A) Scatter plot of the average phenotype score of all embryos in an experiment by the starting concentration of MeHgCl in solution, as measured by DMA. Each triangle point represents one exposure experiment consisting of 10 to 20 embryos. A sigmoidal curve fit of χ2 = 4.36 is shown with its associated R2. N = 32 experiments, with 370 embryos total. (B) Concentration of total Hg detected in embryo dry weight tissue per MeHgCl solution exposure at different embryonic stages as measured by DMA-80. N = 4 experiments, 20 embryos total per dose for stages 4 –8 and 12, and 3 experiments and 15 embryos total per dose for stages 10 and 14–45. Error bars represent standard deviation. (C) Images of embryos at stages 45–46 exposed to indicated amounts of MeHgCl in solution. N = 12 experiments, with 138 total embryos on average per dose.|
|Fig. 4. Expression of early neural patterning and late neural phenotype genes in embryos exposed to varying concentrations of MeHgCl in solution, as assayed by ISH. Stages 23–24 embryos are seen from a dorsal (left) and lateral (right) view probed for sox2 (A–C), en2 (D–F), and delta (G–I). Similarly, shown is the dorsal view of stage 12 embryos probed sox2 (J–M) and delta (N–Q). For neural phenotypes, a lateral view of stage 35 embryos is shown stained for xVGlut (R–U), GAD (V–Y) and xGAT (Z–CC) expression. Dark purple-blue areas indicate positive expression of mRNA. For all images, anterior is on the right, and for lateral views dorsal is up. N = 2 experiments, with the total number of evaluated embryos shown at the lower right of each image. The number of embryos out of the total matching each image is also shown; where counts were evenly split, the embryos most similar to controls are displayed. Abbreviations: e, eye; fb, forebrain; hb, hindbrain; mb, midbrain; olf, olfactory; pg, pineal gland; sc, spinal cord; VII, cranial nerve VII; VIII, cranial nerve VIII; IX, cranial nerve IX. Scale bars represent 1 mm.|
|Fig. 5. Expression pattern of PCNA at several developmental stages during MeHgCl solution exposure, as detected by ISH. Stages 14–15 (A–F), stages 19–20 (G–L), and stages 25–26 (M–R) embryos are seen from a dorsal view. Stages 37–38 embryos (S–X) are shown from a dorsal (left) and lateral (right) view, except for 200 μg/l, which is a featureless ball of cells. All embryos shown are representative near the average phenotype score from exposure at each dose. For all images, anterior is on the right. Dark purple-blue areas indicate positive PCNA expression. N = 2 experiments, with the total number of evaluated embryos shown at the lower right of each image. The number of embryos out of the total matching each image is also shown. (Y) qRT-PCR results of relative PCNA expression in stage 37 embryos at each MeHgCl dose. Values for the δδCт score in each condition have been normalized to the expression level of the 0 μg/l control and are shown using 2− δδCт scale. N = 1 biological experiment pooled from 10 embryos, and 3 technical replicates, for each dose. Scale bars represent 1 mm.|
|Fig. 6. Transverse histological cryosections through stage 37 embryos stained for PCNA expression (blue regions) by ISH. Forebrain, midbrain, hindbrain, and spinal cord sections of control (A–D) and MeHgCl solution exposed (E–H) embryos are shown oriented dorsal on top. Two of the four MeHgCl exposed embryos displayed abnormally reduced or absent forebrain structures as seen in (E). N = 2 experiments, with 4 embryos sectioned from each condition. (I) Neural cell counts using DAPI stain compared between unexposed and MeHgCl exposed embryos averaged over each CNS region. N = 4 experiments, with 7 embryos sectioned from 0 μg/l and 8 from 100 μg/l total. Error bars represent standard deviation. *p = 0.039, **p = 0.0056, ns = not significant as determined by Student's t-test for each region. Abbreviations: e, eye; fb, forebrain; hb, hindbrain; mb, midbrain; olf, olfactory; ot, otic vesicle; pn, pronephros; sc, spinal cord. Scale bar represents 150 μm.|
|Fig. 7. TUNEL staining for cells undergoing apoptosis in whole mount embryos exposed to MeHgCl solution. Shown dorsally are stages 10–12 (A–B), stages 14–15 (C–D), stages 19–20 (E–F), and stages 25–26 embryos (G–H). Stages 37–38 embryos (I–J) are seen from a lateral view. For all images, anterior is on the right. For controls, a low (top) and high (bottom) staining embryo is shown. For the MeHgCl exposed, an average (top) and below average (bottom) ranked embryo is shown for each stage. N = 2 experiments, and 10 embryos total for each. Scale bars represent 1 mm.|
|Fig. 8. Histological sections cut transversely by cryosectioning though stage 37 embryos stained for apoptotic cells (blue dots) by TUNEL assay. Forebrain, midbrain, hindbrain, and spinal cord sections of control (A–D) and MeHgCl solution exposed (E–H) embryos are shown oriented dorsal on top. N = 2 experiments, with 4 embryos sectioned from each concentration. (I) Number of TUNEL positive cells per 100 neural cells compared between unexposed and MeHgCl exposed embryos averaged over each CNS region. N = 2 experiments, with 3 embryos sectioned from each condition. Error bars represent standard deviation. *p = 0.019, **p = 10− 6, ns = not significant as determined by Student's t-test for each region. Abbreviations: e, eye; fb, forebrain; hb, hindbrain; mb, midbrain; olf, olfactory; ot, otic vesicle; pn, pronephros; sc, spinal cord. Scale bar represents 150 μm.|
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