The effects of methylmercury on Notch signaling during embryonic neural development in Drosophila melanogaster

Abstract

Methylmercury (MeHg) is a ubiquitous toxicant that targets the developing fetal nervous system. MeHg interacts with the Notch signaling pathway, a highly-conserved intercellular signaling mechanism required for normal development. Notch signaling is conveyed by activation of the genes in the enhancer of split (E(spl)) locus in Drosophila. We have previously shown that acute high doses of MeHg upregulate several E(spl) genes in Drosophila neural-derived C6 cells. Furthermore, MeHg induction of E(spl) can occur independent of the Notch receptor itself. We now show that MeHg, unlike inorganic mercury (HgCl₂), preferentially upregulates E(spl)mδ and E(spl)mγ in Drosophila C6 cells. This is distinct from Delta ligand-induced Notch signaling in which no induction of E(spl)mδ is seen. MeHg is also seen to specifically upregulate E(spl)mδ in Drosophila embryos where HgCl₂ showed no such effect. Additionally, treatment of embryos with MeHg caused a consistent failure in axonal outgrowth of the intersegmental nerve (ISN). This ISN phenotype was partially replicated by genetic activation of the Notch pathway, but was not replicated by increasing expression of E(spl)mδ. These data suggest a role for Notch signaling and the E(spl)mδ target gene in MeHg toxicity, however, the site of action for E(spl)mδ in this system remains to be elucidated.

Introduction

Methylmercury (MeHg) is a ubiquitous environmental toxin that preferentially targets the developing nervous system. Because of its apparent specificity for neural tissue, signaling pathways in neural development may be important targets in MeHg toxicity. Several studies in both mammalian and invertebrate systems now support the hypothesis that the Notch pathway is a potential target for MeHg. Notch is a fundamental cell-cell signaling pathway that directs cell fate decisions during neurogenesis. Being first elucidated in Drosophila, it is now well understood that signals through Notch receptors cause activation of downstream effectors; those of the enhancer of split [E(spl)] gene locus in flies and the hairy/enhancer of split (HES) genes in mammals (de-la-Concha et al., 1988, Jarriault et al., 1998, Preiss et al., 1988). The E(spl) locus in flies consists of 11 genes in a single 50 kb locus. Seven of these E(spl) genes, E(spl)mδ, E(spl)mγ, E(spl)mβ, E(spl)m3, E(spl)m5, E(spl)m7, and E(spl)m8, are basic helix-loop-helix transcriptional repressors. While different E(spl) genes are known to be preferentially expressed in various developing tissues, manipulations in Drosophila demonstrate that all the E(spl) genes are capable of responding to Notch signals (Jennings et al., 1999, Nellesen et al., 1999, Wech et al., 1999, Wurmbach et al., 1999). Signals at the level of the Notch receptor are propagated by cleavage and activation by members of the ADAM family of metalloproteases. The potential for MeHg to stimulate ADAM activity initially led to the hypothesis that MeHg could ultimately induce Notch signals (Bland and Rand, 2006). This was supported by evidence that E(spl)mγ and E(spl)mβ show a dose-dependent increase in transcription with MeHg applied to Drosophila neural cells in culture (Bland and Rand, 2006). In subsequent studies we have shown that stimulation of E(spl) genes in Drosophila cells by MeHg can occur despite knockdown of Notch receptor expression (Rand et al., 2008). These observations suggest MeHg can act through a more direct mechanism, bypassing the receptor to stimulate transcription of Notch targets.

In this study, using MeHg exposures to Drosophila C6 neural derived cells in culture in addition to exposures of the whole animal at various developmental stages, we confirm a specific action of MeHg toward the E(spl)mδ gene. We also demonstrate that E(spl)mδ, in stark contrast to the other E(spl)s, is not responsive to Notch signals propagated by its cognate ligand, Delta, in the C6 neural cell line, allowing us to elucidate the MeHg specific action on this gene target. A specific effect of MeHg relative to inorganic mercury (HgCl₂) in E(spl)mδ activation is observed in C6 cells and in embryos, however, mercury induction of E(spl)mδ was not seen at later developmental stages. MeHg treated embryos exhibit an overt defect in formation of the intersegmental nerve (ISN). Increasing Notch pathway activity in neurons by driving expression of the Notch intracellular domain (N_ICD) under the control of the pan-neural elav promoter causes a similar defect in ISN outgrowth, however driving expression of E(spl)mδ in neurons did not elicit an ISN phenotype.

Our findings indicate that MeHg specifically effects E(spl)mδ in vitro and in vivo during Drosophila embryogenesis. Our data shows specificity in gene activation by MeHg compared to other stressors, such as HgCl₂, highlighting the potential for E(spl)mδ to mediate some MeHg-induced changes in developmental signaling in the embryo. However, neuron-specific expression of E(spl)mδ did not replicate a characteristic ISN MeHg phenotype, which could be partially replicated with neuron-specific Notch activation. These results point to novel non-canonical Notch pathway mechanisms that contribute to MeHg toxicity in the embryonic nervous system.