Neurodevelopmental Defects in Zebrafish (Danio rerio) at Environmentally Relevant Dioxin (TCDD) Concentrations

Adrian Hill*,1, C. Vyvyan Howard{dagger}, Uwe Strahle{ddagger} and Andrew Cossins*

* Laboratory for Environmental Gene Regulation, School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool Merseyside L69 7ZB, United Kingdom; {dagger} Toxico-Pathology Research Group, Department of Human Anatomy and Cell Biology, Ashton Street Building, University of Liverpool L69 3PX, U.K.; and {ddagger} IGBMC BP10142, 1 rue Laurent Fries, 67404 Illkirch, France

Received July 7, 2003; accepted September 8, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Persistent ecotoxicants, such as dioxin and PCBs, are thought to pose one of the greatest threats to public and ecological health in the industrial world. These compounds cause a range of macroscopic malformations, particularly to the craniofacial apparatus and cardiovascular system during vertebrate development. However, little is known about microscopic effects, especially on the sensitive early life stages or on the molecular basis of developmental neurotoxicity. Using zebrafish (Danio rerio), we have explored neurological deficits caused by early-life exposure to environmentally relevant concentrations of dioxin. We show, using a quantitative stereological technique, that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) substantially reduces the capacity for embryonic brain development, causing a 30% reduction in total neuronal number in the 168-h larval brain. Using transgenic GFP-expressing zebrafish lines, we link this to decreased expression of key developmentally regulated genes, namely neurogenin and sonic hedgehog. This disruption of neuronal development provides the basis for understanding the neurotoxic effects of these compounds.

Key Words: neurotoxicity; TCDD; zebrafish; embryo; sonic hedgehog; neurogenin.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes a variety of detrimental effects in diverse animal taxa. Fish are the most sensitive vertebrates to TCDD, especially during early developmental stages (Peterson et al., 1993Go) making them useful models for exploring perturbations to normal embryonic development. Previous studies on trout, zebrafish, and medaka, using polychlorinated dibenzodioxin, have almost exclusively characterized macroscopic effects such as the incidence of craniofacial malformations, reduced blood flow, impaired swim bladder inflation, and edema of the yolk sac and pericardium (Elonen et al., 1998Go; Henry et al., 1997Go; Teraoka et al., 2002Go). Few studies, however, have investigated the neurotoxic effects of dioxin upon brain formation and development during embryogenesis. Many of these macroscopic effects are determined at high doses of dioxin where effects are clearly expressed and thus easy to visualize. There is much less information on the more subtle effects on tissue structure and formation that might occur at lower dioxin doses, many of which require more quantitative and sensitive methods for detection.

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., 2002Go) and ARNT2 (Tanguay et al., 2000Go) 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., 2000Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dioxin exposure.
Groups of newly fertilized wild-type (AB strain) eggs collected within 2 h of spawning were exposed at the 8-cell stage, in 10 ml of tank water, to 2,3,7,8-tetrachlorodibenzo-p-dioxin (Greyhound Chromatography, U.K.) dissolved in acetone, or to tank water with 0.1% acetone (vehicle control), for a 1-h pulse exposure. Eggs used for the stereological analysis of neuronal density and brain volume were exposed to <=120 ppt (ng/l) TCDD in 20 ppt increments. This range in TCDD concentration was previously determined to include doses just above the minimum concentration required to evoke macroscopic signs of TCDD toxicity such as yolk sac and pericardial edema (data not shown). For assays of apoptosis, eggs were exposed to 100 ppt TCDD. Other techniques were intrinsically less sensitive so higher concentrations of dioxin were employed in order to observe effects. These include the exposure of eggs to 500 ppt TCDD for riboprobe labeling experiments, and to <=400 ppt TCDD for in situ hybridization experiments. After washing, the eggs were incubated in dioxin-free tank water at 27.5 ± 1°C as previously described (Westerfield, 1995Go).

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., 2002Go). 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., 1990Go). 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., 2003Go; Blader et al., 2003Go). The promoter gene sequence for neurogenin 1, neurog1 (-8.4 neurog1:gfp; Blader et al., 2003Go) and sonic hedgehog, shh (-2.2shh:gfpABC; Albert et al., 2003Go) 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)Go, 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)Go. 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stereological Assessment of Brain Tissue
As dioxin is known to cause a generalized retardation of growth, it was necessary to separate this from more specific effects upon brain development. Figure 1Go compares the effects of different concentrations of TCDD upon the brain volumes of larvae of different sizes. This shows for each treatment group that brain volume is a linear function of body size (r2 values: acetone control = 0.7014, 100 ppt TCDD = 0.6336, 120 TCDD ppt = 0.6364), allowing the effects of body size to be corrected in comparing brain volumes between treatment groups. Figure 1Go also shows that increasing TCDD concentration is linked to a shift of brain volume to smaller values for animals of a given size, demonstrating the body-size independence of the effect. Multiplying the brain volume by the measured neuron density provides an estimate of total neuronal number of neurons in the brain (ANOVA, p = 0.001) between the exposed larvae (those treated with 100 ppt TCDD: 66274.8 ± 6411.5 neurons standard deviation (sd); 120 ppt TCDD: 55382.6 ± 5444.2 sd) and vehicle controls (78048.6 ± 5741.7 sd) of similar body volume. This reached a mean decrease of 15% (Dunnett’s p > 0.972) and 29% (Dunnett’s p > 0.999) for the 100 ppt and 120 ppt TCDD groups, respectively. Thus when exposed groups of larvae were compared to those of the controls at 168 hpf, a significant neuronal deficit was caused by exposure to dioxin doses >=100 ppt (ng/l).



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FIG. 1. Dioxin induced a decrease in brain volume in larvae of similar body size at 168 hours post fertilization (hpf). The brain and body volumes for individual larvae of each dosage group—acetone control, 100 ppt TCDD, and 120 ppt TCDD—are shown together with the line of best fit for each data set. Body volumes shown are with the dioxin-induced volume of edema subtracted. Compared to the controls, decreases in mean brain volume for the 100-ppt and 120-ppt dosage groups were significant (Dunnett’s p > 0.95).

 
Effects of Dioxin on Neurogenic Gene Expression
Examination of neurog1:gfp transgenic larvae treated with acetone and TCDD revealed no difference in expression pattern at 24 hpf (Fig. 2Go). However, a visual assessment of pseudocolor (computer-aided enhancement of GFP fluorescence intensity) showed a decrease in neurog1:gfp fluorescence intensity at 48 hpf (Fig. 3Go) caused by doses >=100 ppt TCDD. We have sought to demonstrate changes in neurog1:gfp expression by quantitatively comparing the integrated fluorescence signal from different treatment groups. Differences between mean expression (integrated intensity per unit area of the 2-D confocal projection) of control groups and those exposed to dioxin were evident in a dose-dependent manner. At 48 hpf, differences were observed in the hindbrain of larvae treated at the 400-ppt level (ANOVA, p = 0.014). The mean decrease of integrated fluorescence intensity compared to the controls was 28.1% (Dunnett’s p = 0.998). At 96 hpf, differences in mean integrated fluorescence of neurog1:gfp between the controls and larvae exposed to 140 ppt TCDD were highly significant for the forebrain, midbrain, and whole brain (ANOVA, p = 0.011, p = 0.002, and p = 0.001, respectively). Decreases at this age reached 22.9, 41.3, 19.7, and 29.7% for the forebrain, midbrain, hindbrain, and whole-brain regions, respectively (Fig. 4AGo).



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FIG. 2. Neurogenin expression in the 24-hpf zebrafish brain is unaffected by dioxin exposure. Standard fluorescence (A, C) and image-enhanced pseudocolor (B, D) of neurog1:gfp expression in control embryos (A, B) and embryos exposed to 400 ppt TCDD (C, D) at 24 hpf.

 


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FIG. 3. Neurogenin expression in the brain is decreased by 48 hpf in TCDD-treated embryos. Expression patterns of transgenic zebrafish in the forebrain (F), midbrain (M), and hindbrain (H) at 48 hpf were obtained using a Zeiss LSM 510 confocal scanning fluorescence microscope. Neurog1:gfp expression in the head region, viewed from the side (left) and dorsally (right) as image-enhanced fluorescence pseudocolor, indicated fluorescence intensity (Kinetic Imaging software) in control larvae (A) and larvae treated with 200 ppt TCDD (B) and 400 ppt TCDD (C), demonstrating that dioxin decreased the amount of neurog1 expressed in the zebrafish embryonic brain. Anteriors of larvae are positioned at the base of the image.

 


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FIG. 4. Dioxin causes a decrease in neurogenin 1 expression throughout the brain and a reduction in sonic hedgehog in the hypothalamus and eye in exposed larvae. In larvae at 96 hpf, a decrease in mean integrated fluorescence intensity per µm of neurog1:gfp expression in different regions of the brain (A) and shh:gfp expression in the hypothalamus and eye (B) indicate that doses of TCDD >=100 ppt affect the neurogenesis pathway. Asterisks indicate a significant difference in GFP fluorescence intensity compared to that of controls (Dunnett’s, p > 0.95).

 
To establish whether TCDD affected more discrete populations of cells, another gene Sonic hedgehog, (shh), was selected for study. shh:gfp transgenic zebrafish were used because shh is an important regulator of cell differentiation and proliferation in the central nervous system (Albert et al., 2003Go). Moreover, it is expressed in the hypothalamus, the zona limitans intrathalamica, the ventral midbrain and hindbrain, as well as in the eye, the retina of which is widely used as a model for studying development of the central nervous system. Quantitative assessment of integrated fluorescence expressed in each larvae with the shh:gfp transgene revealed that shh was also downregulated by dioxin exposure. This was evident at significant levels (ANOVA, p = 0.038) in the hypothalamus from 48 hpf when a mean decrease in shh:gfp expression of 7.8% was observed between controls and larvae exposed to 200 ppt TCDD. At 96 hpf (Fig. 5Go), larvae differences in integrated fluorescence intensity were observed at both the 100 and 400 ppt TCDD dosage level (ANOVA, p = 0.05) with a 25 and 26.8% mean decrease, respectively, compared to control larvae (Dunnett’s p = 0.990 and 0.998, respectively; Fig. 4BGo). In addition to the change in brain shh expression, 96-hpf larvae exhibited a decrease in the mean integrated fluorescence intensity in the eye (ANOVA, p = 0.033) of 14.9 and 14.7% for the 100- and 400-ppt dosage groups, respectively (Dunnett’s = 0.995 and = 0.999; Fig. 4BGo).



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FIG. 5. Sonic hedgehog:gfp expression in the head region, viewed anteriorly at 96 hpf by confocal microscopy of GFP fluorescence in control larvae (A) and larvae treated with 100 ppt TCDD (B). Expression patterns are distorted by TCDD exposure, and fluorescence intensity is reduced. Abbreviations: hypothalamus, H; and optic nerve, O.

 
A change in the shh:GFP expression pattern in the brain (Fig. 5Go) was linked to a change in morphology caused by the typical signs of TCDD toxicity, retardation of growth and craniofacial malformations. Although the normal morphological landmarks, such as the midbrain-hindbrain boundary and optic tectum, were all present, the lack of jaw extension and lack of the normal widening of the head in exposed larvae caused the brain to be contorted compared to controls, especially with regard to the position of the optic nerves. As the hindbrain morphological development and the process of metamerization appeared to be unaffected by TCDD exposure, apart from the retardation of growth, the regions of expression in this tissue were not as contorted. Changes in gene expression in the affected tissues may also be linked to a lack of blood nutrients due to slowed blood flow (Dong et al., 2002; Henry et al., 1997Go).

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. 6Go), 48 (Fig. 7Go), 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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FIG. 6. The expression patterns of numerous genes involved in the development of the eyes and brain appear unaffected by dioxin exposure. Whole mount in situ hybridization in control embryos (A, C, E, G, I, K, M, O, Q, S) and embryos exposed to 500 ppt TCDD (B, D, F, H, J, L, N, P, R, T) of the riboprobes for ashb (A, B), neurod (C, D), isl1 (E, F), wnt1 (G, H), pax2a (I, J), pax6a (K, L), egr2b (M, N), rtk1 (O, P), dbx1b (Q, R) and dlx3b (S, T) show no differences in expression pattern at 24 hpf. All larvae are shown in lateral view with the head facing to the left, except those for pax6a and dlx3b, which are shown in the dorsal view.

 


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FIG. 7. At 48 hpf, the in situ expression patterns of several genes appeared unaffected in the brains of embryos exposed to dioxin. Expression patterns of isl1 (A, B), pax2a (C, D), pax6a (E, F) and shh (G, H) are shown in the side view with the head facing left.

 
Assessment of Apoptosis during Early Development
Using the vital dye acridine orange to stain pyknotic cells, a severe inhibition in programmed cell death (apoptosis) was exhibited in larvae treated with 100 ppt TCDD as demonstrated by the absence of acridine orange-positive cells (Fig. 8Go). This was evident from spawning to 2.5 days postfertilization, at which time the distribution of fluorescent cells increased to comparable levels observed in the controls. At 28 hpf, pyknotic cells were evident in clusters and single cells throughout the brain of control larvae (Fig. 8AGo), but were especially condensed in the forebrain.



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FIG. 8. Apoptosis in the zebrafish embryo viewed by confocal microscopy of fluorescent acridine orange stain is affected by TCDD exposure. Compared to the side view of the control embryos (head, A), a complete inhibition of apoptosis was observed at 28 hpf in embryos treated with 100 ppt TCDD (head; B). By 48 hpf, a few acridine orange-positive cells were observed in the tail of exposed embryos (D) compared to controls (C), and by 80 hpf, levels of apoptosis in the brain of control embryos (ventral view of head, E) were similar to, or in excess of levels observed in exposed larvae (corresponding view; F).

 
Another site of high fluorescence and therefore apoptosis was the eye. In addition numerous isolated apoptotic cells were present in the tail. These observations of control larvae showed similar temporal and spatial distributions of apoptotic cells as previously documented (Cole and Ross, 2001Go). In contrast, almost all larvae exposed to TCDD exhibited no fluorescence anywhere in the whole body at 28 hpf (Fig. 8BGo). Two larvae out of eight showed a total of one or two singular apoptotic cells, none of which were in the brain. At 48 hpf, a few more clusters of apoptotic cells were present in the tail and trunk regions, but none were evident in the head (Figs 8C Goand 8DGo). At around 58 hpf, levels of apoptosis in the brain had increased in exposed larvae and appeared more extensive by 80 hpf (Figs 8E Goand 8FGo) than controls.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using the zebrafish (Danio rerio) vertebrate model, we show that early embryonic exposure to dioxin causes severe neurological deficits in subsequent developmental stages.

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, 2002Go), 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)Go 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 0–256 and 5–495 pg/g muscle wet-weight, respectively, and found TCDD concentrations of 3–16 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, 1994Go), 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., 1993Go). 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., 2000Go), the otic system, spinal cord, and specific interneurons of the hindbrain (pax2a: Pfeffer et al., 1998Go), the eye (pax2a and pax6a: Pfeffer et al., 1998Go; Ziman et al., 2001Go) and hindbrain (egr2b, dbx1a, rtk1, pax2a and dlx3b: Fjose et al., 1994Go; Makori et al., 1999Go; Schier et al., 1996).

13-cis-Retinoic acid (RA)-induced craniofacial malformations (Helms et al., 1997Go) 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., 1999Go), 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., 1993Go). 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., 2001Go). 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, 1992Go), 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., 1988Go) and use of agent orange (Khan et al.,1988Go). 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., 1997Go) as well as through placental transport (Jacobson et al., 1984Go) 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, 2002Go). In addition to the known teratogenic relationship between dioxins and dioxin-like substances and cleft palate (Gordon and Shy, 1981Go), neurological deficiencies such as delayed motor function and altered cognitive function associated with dioxin exposure (Chen et al., 1994Go; Gladen et al., 1988Go; Huisman et al., 1995Go; Patandin et al., 1999Go) 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.


    ACKNOWLEDGMENTS
 
A.J.H. was supported by a postgraduate studentship, and A.R.C. by research grants, both from the Natural Environment Research Council (U.K.). U.S. was supported by CNRS, INSERM, ULP, ARC, and AFM.


    NOTES
 
1 To whom correspondence should be directed at the present address: 5206 Rennebohm Hall, School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI 53705. E-mail: ajhill{at}pharmacy.wisc.edu. Back


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