Decreased axonal density and altered expression profiles of axonal guidance genes underlying lead (Pb) neurodevelopmental toxicity at early embryonic stages in the zebrafish

Abstract

Previous studies have reported that environmental lead (Pb) exposure can result in neurological alterations in children leading to reduced IQ, attention deficit hyperactivity disorder, and diminished reading and learning abilities. However, the specific alterations in neurodevelopmental morphology and the underlying genetic mechanisms of these alterations have not yet been thoroughly defined. To investigate alterations in neurologic morphology and test the hypothesis that developmental Pb neurotoxicity is partially mediated through alterations in neuronal growth and transport function of axons, the changes of specific axon tracts in the embryonic zebrafish brain were observed with anti-acetylated α-tubulin staining at several developmental time points through 36 hours post fertilization (hpf). In addition, the role of a subset of axonogenesis-related genes including shha, epha4b, netrin1b, netrin2, and noi were investigated with real-time quantitative PCR (qPCR). Pb treatment resulted in decreased axonal density at 18, 20, and 24 hpf for specific axon tracts in the midbrain and forebrain. These observations corresponded to an observed down-regulation of shha and epha4b at 14 and 16 hpf, respectively. The axonal density in Pb exposed individuals at later stages (30 and 36 hpf) was not significantly different from controls. An overexpression of netrin2 at these two developmental stages suggests a novel role for this gene in regulating axonal density specific to Pb neurotoxicity. Although no significant differences in axonal density was observed in the two later developmental stages, further studies are needed to determine if the morphologic alterations observed at the earlier stages will have lasting functional impacts.

Zebrafish as a Model for Developmental Neurotoxicity Assessment: The Application of the Zebrafish in Defining the Effects of Arsenic, Methylmercury, or Lead on Early Neurodevelopment

“…To facilitate the application of the zebrafish model in DNT testing, further studies need to be conducted on exposure kinetics of various substances to determine exposure doses per embryo or larva. A few studies have started to include this analysis (e.g., [69,70]), but more work is needed in this area to understand dose and exposure kinetics. In addition, chorionation or dechorionation status at the time of chemical exposure may also affect the degree of chemical absorption by the embryo.…”

“…The genetic mechanisms underlying this Pb-induced axonal density decrease were investigated by measuring quantitative expression of genes involved in axon guidance in embryos exposed to 100 ppb Pb through 14-36 hpf. The qPCR analysis revealed that expression of sonic hedgehog a and ephrin type-A receptor 4b were significantly down-regulated at 14 and 16 hpf, respectively [70]. On the other hand, netrin2 expression increased significantly at 30 and 36 hpf, suggesting the involvement of netrin2 in regulating axonal growth in response to Pb neurotoxicity at early developmental stages [70].…”

“…The zebrafish presents numerous strengths as an in vivo test model including ex utero fertilization and transparency of embryos and early larvae, enabling microscopic observation through early developmental stages. The ex utero embryonic development also eases the determination of doses of exposed chemicals at all embryonic stages, providing explanation of exposure kinetics of chemicals of interest [67][68][69][70]. Moreover, the rapid growth and high fecundity of zebrafish facilitate higher throughput toxicity testing of multiple chemicals.…”

“…In an additional follow-up study, the effect of Pb exposure on axonal growth in the developing zebrafish was studied following exposure to 100 ppb Pb acetate at several time points between 18-36 hpf by α-AT staining and showed a significant decrease in axonal density in Pb exposed embryos at 18, 20, or 24 hpf [70]. The genetic mechanisms underlying this Pb-induced axonal density decrease were investigated by measuring quantitative expression of genes involved in axon guidance in embryos exposed to 100 ppb Pb through 14-36 hpf.…”

Novel dose-dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity

“…The zebrafish is becoming an increasingly popular model in developmental neurotoxicity studies (e.g., Chen et al, 2012; Peterson et al, 2011, 2013; Rice et al, 2011; Zhang et al, 2011). There are considerable strengths in utilizing the zebrafish model to assess developmental neurotoxicity including ex utero embryonic development, high fecundity rate therefore increasing sample size and biological replicates, a transparent chorion allowing for easy observation of gross morphological changes, and a fully sequenced genome which exhibits high homology to humans (Barbazuk et al, 2000; de Esch et al, 2012; Howe et al, 2013).…”