We have previously demonstrated an ability of MeHg to induce E(spl) gene expression in cultured Drosophila
C6 cells (Rand et al., 2008
). In this previous study EDTA was used as a proxy to invoke Notch signaling in C6 cells for comparative effects on E(spl) expression. Since notable differences between EDTA-induced and MeHg-induced E(spl) activation was observed, we wished to further validate these effects with respect to Notch signaling. To examine Notch signaling explicitly we co-cultured the C6 cells on fixed preparations of cells expressing Delta, the endogenous ligand of the Notch receptor, in a previously established assay in our laboratory (Delwig and Rand, 2008
). We then analyzed E(spl) expression exclusively in the Notch expressing cells via qRT-PCR (). This treatment caused the greatest induction of E(spl)m
β and E(spl)m3
(>20-fold), and a nearly 15-fold induction of E(spl)m
γ. In contrast, E(spl)m
δ was not upregulated by Delta induced Notch signaling. Overall, Delta induced Notch signaling under these conditions gives a similar pattern of E(spl) induction as was seen with EDTA earlier (Rand et al., 2008
) indicating this profile is representative of Notch signaling in this cell line.
E(spl) gene expression in Delta ligand induced Notch signaling
We next sought to compare MeHg effects with inorganic mercury to determine if the E(spl) activation profile of MeHg is unique or shares properties with other mercurials. We first determined levels of toxicity of MeHg and HgCl2 toward C6 cells. Cell viability subsequent to MeHg and HgCl2 exposures was determined using dual calcein/ethidium staining (). A dose dependent decrease in cell viability was observed with MeHg, which proved more potent than HgCl2. MeHg exhibited an approximate 20% cell death (80% viability) at 4μM and an approximate 50% reduction in viability at 20μM. HgCl2 proved weaker showing an approximate 20% reduced viability at 20μM and 50% reduced viability with 100μM HgCl2. Similar results were obtained using alternative determinations with Trypan Blue reagent (Data not shown). These doses were implemented in subsequent assays of acute exposure effects on E(spl) activation.
Cell viability of Drosophila neural-derived cells after mercurial treatment
With MeHg treatment of C6 cells E(spl)mδ, E(spl)mγ, and E(spl)m7 showed the greatest fold-induction (7 to 12-fold, ) of six representative E(spl) genes spanning the E(spl) locus. E(spl)mβ and E(spl)m3 showed less substantial increases (less than 4-fold) with MeHg treatment, while E(spl)m2 approached a 6-fold induction. In contrast, cells treated with HgCl2 showed less than 3-fold response in E(spl)mδ and E(spl)mγ, and less than 5-fold induction in E(spl)m7 (). E(spl)m2 responded to HgCl2 treatment with a nearly 9-fold change with 100μM MeHg (). These data show a differential response of individual genes in the E(spl) locus with MeHg versus HgCl2 exposure.
E(spl) gene induction by mercurial treatment
The apparent unique effect of MeHg on E(spl) gene expression in vitro
prompted us to investigate similar effects in vivo
. We have previously established an ability to dose Drosophila
embryos with MeHg cultured in vitro (Rand et al., 2009
). This methodology is summarized in . The method takes advantage of the unique property that fly embryos denuded of their outer chorion layers of the eggshell are permeable to MeHg and are also able to continue development suspended in a defined culture media (see methods and ). Using this technique we evaluated the dose-response of embryos to MeHg by assaying gene expression using qRT-PCR and monitoring the response of a ubiquitous stress response gene, Hsp70
. The Hsp70 Bc
gene showed a robust increase in expression with increasing levels of MeHg, confirming the entry of the MeHg in embryonic tissues (). In parallel we probed E(spl)m
δ gene expression which was seen to increase across all concentrations of MeHg. A more than seven-fold increase in E(spl)m
δ was seen at 20μM MeHg, which appeared to be sustained at 50μM MeHg. From these data we chose to treat embryos with 50μM MeHg in subsequent analyses to ensure we were above a threshold in effect.
Dose response of E(spl) mδ in Drosophila embryos after MeHg treatment
To assess the level of specificity with which MeHg acts on embryonic tissues we again compared E(spl) expression response to MeHg versus HgCl2 treatment (). After MeHg, treatment (50μM) E(spl)mδ consistently showed greater than three-fold upregulation in embryos across several trials. In contrast, none of the other E(spl) genes assayed showed a response to MeHg in embryos treated in vitro. In addition, Notch showed no change in expression in response to MeHg, indicating that increases in E(spl)mδ could not stem from increased receptor expression. An induction of two Hsp70 genes Hsp70Ab and Hsp70Bc was observed for MeHg, again confirming entry of the toxicant into the embryos. Treatment of embryos with 1mM HgCl2 did not cause any increase in E(spl) gene expression. In contrast, a modest decrease was seen in levels across all the E(spl) genes and Notch after HgCl2 treatment. Upregulation of Hsp70 genes after HgCl2 treatment indicated that the dose of HgCl2 used showed a similar degree of entry and overall toxic insult to that of MeHg. These data indicate that MeHg acts selectively on E(spl)mδ transcription in Drosophila embryos.
E(spl) gene induction in Drosophila embryos after mercurial treatment
E(spl) gene expression is known to change over the course of embryogenesis (Tweedie et al., 2009
). Recent data from gene expression arrays performed within the large scale ModEncode project (Tweedie et al., 2009
) and publically available on Flybase (Graveley et al.) permit a comprehensive analysis of developmental expression of the E(spl) gene during normal embryogenesis. Transcript levels of E(spl)m
γ, and E(spl)m7
show a similar profile as Notch, which shows a peak of expression at 6-8 hours of development after egg laying (). In contrast, E(spl)m3
shows peak expression discernibly later, peaking at 8-10 hours AEL. The bell-shaped expression of the E(spl) genes prompted us to test whether the effect of MeHg on increasing E(spl)m
δ in embryos was simply due to a developmental delay and shift in peak of gene expression versus an ectopic induction of gene expression. To achieve this we incubated batches of developmentally staged embryos with or without MeHg for various treatment intervals, and compared E(spl) expression via qRT-PCR. In untreated embryos the overall profile of E(spl) expression showed the characteristic increase followed by a decrease over the course of embryogenesis. For E(spl)m
γ, and E(spl)m7
a peak of expression between 6-8 hours after egg laying (AEL) was observed (). For E(spl)m3
this peak was seen between 8-10 hours AEL. Notch
expression showed a gradual decline over the course of embryogenesis. With MeHg treatment, E(spl)m
δ showed higher expression at each time point after 6hr AEL compared to untreated embryos, with more than 2-fold higher expression at the 8-10hr interval. Peak expression remained at the 6-8 hour AEL interval with MeHg, indicating the developmental delay was not substantial at this time point. Higher expression due to MeHg was not consistently observed for E(spl)m
γ and E(spl)m3
, while E(spl)m7
did show modest increases at time points after 8hrs AEL with MeHg treatment. Notch
showed no consistent difference in expression due to MeHg treatment over these developmental periods (). Hsp70 Bc
, like E(spl)m
δ, showed increased expression due to MeHg treatment at every time point, confirming the access of MeHg to embryonic tissues at all stages.
Relative gene expression levels of select E(spl) genes and Notch during embryonic development
Response of E(spl) genes in Drosophila embryos after MeHg treatment over the course of development
Observing that E(spl)m
δ is upregulated in embryos after MeHg treatment we investigated whether or not the MeHg effect was penetrant at later developmental stages. First instar (L1) larvae were cultured on food containing 15μM MeHg and harvested for qRT-PCR after reaching the wandering third instar (L3) stage. Previous analyses have demonstrated that treatment of larvae with 15μM MeHg shows similar toxicity to 50μM treatments of embryos (Mahapatra et al., 2010
; Rand et al., 2009
). We then examined global transcript levels of E(spl)s and Notch
from whole larval extracts using qRT-PCR (). No change in expression due to MeHg in E(spl)m
δ or any E(spl) in the larvae was observed. These data suggest that Drosophila
tissues at later developmental stages than the embryo are refractory to MeHg induced expression of E(spl).
E(spl) gene induction in Drosophila larvae after MeHg treatment
To further test whether the effect of MeHg on E(spl)mδ expression is specific to the embryo we tested E(spl) expression responses to MeHg in adult flies. Adult Drosophila were cultured for three days on food containing various concentrations of MeHg up to 100μM. RNA transcript levels in extracts prepared from isolated heads were assayed by qRT-PCR. We opted to examine this tissue since Notch activity and E(spl) expression is a strong determinant in neural tissues and the fly head is rich in brain tissue. We determined that no consistent changes in E(spl)mδ, or any of the E(spl) genes examined, were seen with MeHg treatments (data not shown). Altogether, the data from larval and adult assays indicate that the global effect of MeHg on E(spl)mδ expression is specific to embryos.
We next turned to examining the embryonic nervous system for phenotypes that characteristically reflected MeHg insult to development. Our previous studies have identified several features in the embryo CNS and PNS that reflect compromised development with MeHg exposure (Rand et al., 2009
). Notably, neurite outgrowth of CNS and PNS axons visualized in the lateral field of the late stage embryo has been seen to be compromised (Rand et al., 2009
). Using an antibody specific to the ISN, a bundle of four axons of central motor neurons, we observed that these axons frequently failed to develop properly in MeHg treated embryos (). This phenotype of MeHg exposure presents as stunted or misguided ISNs in treated embryos () and is easily scored by the growth of axons relative to the position of elav-positive PNS neurons in the lateral field (). Interestingly, previous studies have identified a role for Notch signaling in the appropriate projection of the ISN neuron (Giniger et al., 1993
MeHg treatment causes axonal disruption in embryos
We therefore set out to test whether genetic manipulations of Notch in general, and E(spl)mδ in particular, could mirror effects of MeHg on ISN development. We first examined the effect of ubiquitous activation of Notch in post mitotic neurons by driving expression of NICD under control of the pan-neural elav promoter using the GAL4/UAS system (see methods). Ectopic expression of NICD in neurons could be detected with an antibody to the NICD () and by qRT-PCR. In general, the potent activity of Notch was evident by an overall failure of a substantial number of embryos to develop to late stages. Of those that were able to develop to late stage a common feature was seen in a stunted outgrowth of the ISN () akin to that seen with MeHg treatment. As E(spl)mδ was the most consistent E(spl) responder to MeHg we next determined if E(spl)mδ overexpression by the elav promoter could elicit an analogous MeHg-like ISN phenotype. Despite unambiguous overexpression of E(spl)mδ in elav-GAL4>UAS-E(Spl)mδ embryos (as determined by qRT-PCR, as no E(spl)mδ antibodies are available) no apparent phenotype in ISN development was observed ().
Notch pathway activation in neurons disrupts nerve outgrowth in embryos that is not induced with E(spl)mδ overexpression