* Laboratory for Environmental Gene Regulation, School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool Merseyside L69 7ZB, United Kingdom;
Toxico-Pathology Research Group, Department of Human Anatomy and Cell Biology, Ashton Street Building, University of Liverpool L69 3PX, U.K.; and
IGBMC BP10142, 1 rue Laurent Fries, 67404 Illkirch, France
Received July 7, 2003; accepted September 8, 2003
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ABSTRACT |
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Key Words: neurotoxicity; TCDD; zebrafish; embryo; sonic hedgehog; neurogenin.
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INTRODUCTION |
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There is also limited information on the effects of TCDD at the level of gene expression. In the nervous system, TCDD-induced effects may be exerted through a ligand-activated transcription factor known as the aryl hydrocarbon receptor (AhR) together with the aryl hydrocarbon nuclear translocator (ARNT), since increased expression of AhR2 (Andreasen et al., 2002) and ARNT2 (Tanguay et al., 2000
) have been found in the zebrafish brain after dioxin exposure. In addition to zebrafish, AhR and ARNT have been identified in the olfactory bulb, cerebral and cerebellar cortices, and the hippocampus of exposed rats (Huang et al., 2000
). This suggests that TCDD could have significant effects on discrete neuronal populations of the vertebrate brain, though how this relates to disturbed development of the brain or its subsequent function is not known. Moreover, the effects of dioxin toxicity on developmentally regulated genes that underpin the brain development are not known.
In the current study, we have investigated dioxin-induced effects on neurogenesis and brain development in the embryonic zebrafish (Danio rerio), using quantitative and semiquantitative microscopic techniques. First, we use an unbiased, three-dimensional stereological reconstruction technique to determine brain volume and neuronal density in embryos to thereby quantify the effects of dioxin doses on both measurements over developmental time. Second, using transgenic GFP-expressing strains of zebrafish and in situ hybridization techniques, we explore the extent to which the expression of key neurodevelopmental genes is spatially disturbed by dioxin exposure. We demonstrate an effect of TCDD upon the development of brain volume through reduction in the number of brain neurons, and we correlate this with disturbances to the expression of neurogenin and sonic hedgehog genes.
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MATERIALS AND METHODS |
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Stereological assessment of brain volume and neuron density.
Larvae were collected at 168 hpf (hours post-fertilization), euthanized, and embedded in Technovit 7100 resin (Kulzer Gmbh & Co., supplied by TAAB Laboratories Equipment Ltd., Berks., U.K.) as previously reported (Hill et al., 2002). Serial 25-µm sections were stained with Giemsa solution (azure, eosin, methylene blue). Volumes of brain tissue were estimated using the Cavaleri method and assessments of brain neuronal density were performed using the optical dissector technique (Braendgaard et al., 1990
). Brain volumes were multiplied by the neuron density to obtain an estimate for total number of neurons in each brain. To avoid bias when comparing these measurements between larvae of similar body size, the volume of dioxin-induced yolk-sac edema (fluid-filled space surrounding the yolk), calculated using the same technique, was subtracted from the overall body volume of both control and exposed larvae.
Transgenic study.
Generation of GFP transgenic zebrafish lines was reported previously (Albert et al., 2003; Blader et al., 2003
). The promoter gene sequence for neurogenin 1, neurog1 (-8.4 neurog1:gfp; Blader et al., 2003
) and sonic hedgehog, shh (-2.2shh:gfpABC; Albert et al., 2003
) drive spatially and temporally correct expression in the central nervous system. Eggs exposed to dioxin were obtained from out-crossed stable transgenic lines maintained in Strasbourg. Larvae were removed at daily intervals and anesthetized in a 1/7000 weight by volume dilution of MS222 in embryo medium, to allow visual assessment of GFP expression using a Zeiss LSM 510 confocal scanning fluorescence microscope. A solution of 3% w/v methylcellulose was used to orientate the embryos for correct imaging. As any voxel can be allocated a level of fluorescence (fluorescence units: FUnits, calculated on a proportional basis to the maximum level of fluorescence that can be recorded by the detector), the intensity (I) of any region in a scanned 3-D image can be quantified. This was achieved using Kinetic AQM and Lucida software to display the 3-D image as a 2-D integrated intensity projection (IIP). The IIP is a sum of all the voxel intensities at each focal point throughout the Z stack. These measurements were normalized to integrated fluorescence intensity per mm2. Total integrated fluorescence intensity was quantified for the forebrain, midbrain and hindbrain (neurog1:gfp), and hypothalamus and eye (shh:gfp) in replicate specimens at 48, 72, and 96 hpf.
Whole mount in situ hybridization.
Whole mount in situ hybridization was performed simultaneously for control and TCDD-exposed larvae, essentially as described by Oxtoby and Jowett (1993), using riboprobes to neurod (neurogenic differentiation), pax6a (paired box 6a), pax2a (paired box 6a), egr2b (early growth response 2b), wnt 1, rtk1 (eph-like receptor tyrosine kinase 1), isl1 (Islet 1), ashb (achaete scute homolog b), dbx1a (developing brain homeobox 1a), dlx3b (distal-less homeobox gene 3b), and shh (sonic hedgehog). All riboprobes were labeled with digoxigenin and detected using anti-DIG AP (1:4000) and BCIP/NBT substrate (Sigma, Surrey, U.K.).
Assessment of apoptosis using acridine orange.
Larvae were dechorionated before treatment for 30 min with 5 mg/ml acridine orange (AO) w/v in embryo medium for larvae up to 48 hpf, or 10 µg/ml AO for larvae over 48 hpf, as described by Furutani-Seiki et al. (1996). A three-dimensional image of AO fluorescence in control and TCDD-exposed larvae was produced at various time points, during the first three days of development, with a Zeiss LSM 510 confocal scanning fluorescence microscope to enable the assessment of apoptotic cell distribution.
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RESULTS |
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Whole-mount in situ hybridization was performed for eleven riboprobes of genes involved in brain and eye development and patterning. Expression patterns of the genes neurod, pax 6a, pax2a, egr2b, wnt 1, rtk 1, isl1, ashb, dbx1a, dlx3b, and shh showed no obvious differences in the brain or eye expression patterns between the control larvae and larvae exposed to 500 ppt TCDD at 24 hpf (Fig. 6), 48 (Fig. 7
), or 72 hpf (data not shown). Clear differences in the expression patterns of dlx 3b were observed at 72 hpf in the branchial arches.
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DISCUSSION |
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Dioxin-Induced Deficiencies in Brain Development and Decreased Gene Expression
Quantitative stereological analysis of brain tissue in larvae exposed to TCDD revealed that dioxin substantially reduces the capacity for embryonic brain development, causing a 30% deficiency in total neuronal number in the 168-hpf larval brain. We show that this effect is independent of suppressed growth of the whole body. Altered neuronal development is likely to involve disruption of the developmental cascade of genes involved in neurogenesis. Utilizing transgenic zebrafish lines expressing GFP under the control of key promoters regulating the neural differentiation we demonstrated that TCDD disrupted neurogenesis at the molecular level. These results are consistent with the idea that TCDD retards development of neurological tissue at the 100-ppt TCDD exposure level, a contaminant level commonly found in the environment, by either acting on the transcription of the neural regulator neurog1 (Korzh and Strähle, 2002), or by affecting other genes or essential transcription factors somewhere upstream in the neurogenic cascade. Eye and craniofacial development, disturbed after dioxin exposure may also be attributed through actions on shh. Decreased neurog1 in the brain and shh expression in the hypothalamus and eyes indicate that neurological toxicity caused by dioxin exposure may be mediated through different aberrant gene expression pathways in different populations of neurons.
By using GFP transgenics to explore the temporal and spatial expression of specific genes, it was possible to obtain high-definition, three-dimensional quantitative results that are not possible using conventional techniques such as in situ hybridization. This method is therefore a valuable probe for subtle neurological toxicity testing.
These results may indicate that exposed fish larvae with a decreased neurological capacity could have learning or behavioural disabilities in early development, possibly leading to a lower survival rate through increased predation. Since dioxin is a bioaccumulative chemical, these neurological deficiencies may be more prominent in the fish higher up in the food chain due to the probable maternal transfer of TCDD to the egg. This transfer was demonstrated by Giesy et al.(1999) in salmon and trout in the Great Lakes, where "blue sac" disease (a disease characterized by yolk-sac and pericardial edema), a manifestation of TCDD toxicity, is common. They reported polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) levels of between 0256 and 5495 pg/g muscle wet-weight, respectively, and found TCDD concentrations of 316 pg/g egg in lake trout.
Dioxin Does Not Affect the Structural Morphology of the Brain
Genes having key developmental roles for neurogenesis and/or brain morphology were screened for TCDD-induced disruption using whole mount in situ hybridization. Given the effect on brain size, genes known to be involved in neurogenesis were first investigated. The ashb gene is expressed in the ventral tegmentum, neural retina, telencephalon, diencephalons, epiphysis, spinal cord, and hindbrain early in the neurogenesis pathway (Allende and Weinberg, 1994), whereas neurod and isl1 are activated further downstream. Isl1 is activated after neurod, and is expressed in the following primary neurons, trigeminal ganglion and Rohon-Beard, and in interneurons (Korzh et al., 1993
). No obvious differences in the expression pattern between control and TCDD-treated embryos were observed for these genes up to 72 hpf. This suggests that TCDD does not have a global effect on gene expression, but rather is limited to a few key genes having highly specific effects. As numerous genes are regulated during brain development, further investigations should evaluate the impact of TCDD on a wider representative group of developmental genes. Additionally, as demonstrated by the in situ and transgenic expression patterns of shh, although the appearance of gene expression may be similar in control and TCDD-exposed larvae, quantification of these genes may later show that they were affected.
Besides retardation of growth and the reduction of neurons in the brain, gross anatomical assessments of the eye (rod and cone layers) and the brain (different regions such as the epiphysis, midbrain-hindbrain boundary, telencephalon, and diencephalon) revealed that they were structurally unaffected by dioxin exposure. These observations were supported by the normal gene expression patterns in many regions of the central nervous system in exposed larvae, including the midbrain-hindbrain boundary, an organizing center that controls cerebellum and midbrain development (wnt and pax2: Bouchard et al., 2000), the otic system, spinal cord, and specific interneurons of the hindbrain (pax2a: Pfeffer et al., 1998
), the eye (pax2a and pax6a: Pfeffer et al., 1998
; Ziman et al., 2001
) and hindbrain (egr2b, dbx1a, rtk1, pax2a and dlx3b: Fjose et al., 1994
; Makori et al., 1999
; Schier et al., 1996).
13-cis-Retinoic acid (RA)-induced craniofacial malformations (Helms et al., 1997) are similar to those manifested after TCDD exposure. RA has been shown to cause a reduction in the size of rhombomere (R) R4 and R5, and also, fewer egr2b-expressing post otic neural crest cells (NCC) to migrate from R5 and fewer pax2a-expressing NCC to migrate to the second pharyngeal arch from R4 (Makori et al., 1999
), supporting the possibility that hindbrain disruption is the primary mechanism of RA action. It was, thus, unexpected that the hindbrain development was intact in dioxin-treated embryos, suggesting a mechanism distinct from RA toxicity.
Effects of TCDD on Apoptosis
Acridine orange is a vital dye that selectively stains for apoptotic cells, possibly through biochemical changes present only during programmed cell death (Abrams et al., 1993). A possible TCDD-induced increase in apoptosis, observed in this study after 2.5 days postfertilization, may have contributed to the decreased number of neurons in the brain at 168 hpf. This would therefore support the increase previously reported in the tectal region of the TCDD-exposed zebrafish brain at 60 hpf analyzed using TUNEL staining (Dong et al., 2001
). However, an unexpected result, a severe inhibition in programmed cell death (apoptosis) was exhibited in exposed larvae from spawning to 2.5 days postfertilization. As this inhibition, in theory, would prevent the normal remastering of neuronal tissue by apoptosis, exposure to dioxin could first lead to disorganized and poor signal transduction and loss of brain function. This loss of neuronal patterning could result in the observed increase in apoptosis later in development.
Relevance to Terrestrial Vertebrates
Brain development is a highly complex process involving numerous genes and pathways, and the exact mechanism behind dioxin-induced neurodevelopmental toxicity is yet to be established. However, as genes, receptors and molecular processes within the vertebrates are highly conserved, neurological studies with the zebrafish are likely to be representative for other vertebrates, including mammals. Thus the clear-cut effect of TCDD upon brain neurogenesis and brain growth, perhaps mediated by disruption to the expression of key developmentally regulated genes, suggests that similar effects occur in terrestrial vertebrates, including humans. Humans are regularly exposed to dioxins by the intake of contaminated food (Rappe, 1992), of which meat, milk, and fish tend to be the dominant sources. Populations have been exposed to a greater extent through industrial accidents (Mastroiacovo et al., 1988
) and use of agent orange (Khan et al.,1988
). Teratogenic effects of TCDD are particularly relevant because fetal and infant exposure is common as the transfer of high dioxin levels through lactational exposure (Patandin et al., 1997
) as well as through placental transport (Jacobson et al., 1984
) has been demonstrated. Recent evidence of long-term CNS effects in humans associated with intrauterine dioxin exposure suggested there was a developmental delay of about 1 year in a cohort of 41 children aged between 7 and 12, whose intrauterine and perinatal dioxin dosage had been estimated from their mothers body burdens (ten Tusscher, 2002
). In addition to the known teratogenic relationship between dioxins and dioxin-like substances and cleft palate (Gordon and Shy, 1981
), neurological deficiencies such as delayed motor function and altered cognitive function associated with dioxin exposure (Chen et al., 1994
; Gladen et al., 1988
; Huisman et al., 1995
; Patandin et al., 1999
) are less well understood. The detrimental effects to the neurogenesis pathway and the loss of neurons in exposed zebrafish larvae described in this study provide a potential explanation for subtle deleterious effects observed in terrestrial vertebrates such as prenatally exposed children.
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ACKNOWLEDGMENTS |
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NOTES |
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