* School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705; and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705
Received November 4, 2003; accepted December 19, 2003
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ABSTRACT |
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Key Words: TCDD; dioxin; zebrafish; common cardinal vein; morpholino.
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INTRODUCTION |
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Another way to assess involvement of the AHR in vascular development is by investigating the effects of AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experiments utilizing Ahr-null mice have shown that nearly all signs of TCDD toxicity require the presence of a functional AHR (Fernandez-Salguero et al., 1996). However, assessing vascular development in fetal mice is complex, and repeated observations of the same fetus cannot be made. The ease of observations made possible by transparent zebrafish larvae, combined with transgenic techniques for labeling their vascular endothelial cells with green fluorescent protein (GFP), permits repeated, detailed observations of vascular development to be carried out in the same larva. This is important, as the vascular structures proposed to be affected by TCDD are transient, undergoing rapid changes during the course of development.
The AHR pathway in fish is similar to that in mammals (Hahn, 1998). Therefore, zebrafish can be used to conduct detailed examinations of the role of AHR in vascular development, and these results can then be related to effects seen in higher vertebrates. Many of the key components of the AHR pathway have been sequenced and characterized in zebrafish (Tanguay et al., 1999
; Tanguay et al., 2001; Andreasen et al., 2002a). Zebrafish have two AHRs (AHR1 and AHR2), which are the product of two distinct genes. Although both AHR1 and AHR2 bind to dioxin-responsive elements (DREs), only zebrafish AHR2 specifically binds TCDD and is able to transactivate reporter constructs after TCDD exposure in transient-transfection studies (Tanguay et al., 1999
; Andreasen et al., 2002
). Further, zebrafish ahr2 morpholino protein knockdown studies have shown that blocking the expression of zebrafish AHR2 is sufficient to block most signs of TCDD developmental toxicity in zebrafish larvae through 96 h post fertilization (hpf) (Prasch et al., 2003
). AHR nuclear translocator 2b (ARNT2b) is the only known form of zebrafish ARNT that is active with a DRE-driven reporter (Tanguay et al., 2000
).
Zebrafish respond to TCDD with the induction of cytochrome P4501A (CYP1A) (Tanguay et al., 1999), and the TCDD dose required to induce CYP1A in whole zebrafish larvae is similar to that which produces developmental toxicity (Henry et al., 1997
). Evidence suggests that the vascular endothelium is extremely sensitive to induction of CYP1A by TCDD, and this induction can be correlated to apoptosis of endothelial cells in medaka embryos (Cantrell et al., 1998
) and to mortality associated with blue sac syndrome in lake trout sac fry (Guiney et al., 1997
). Further, yolk sac edema fluid from lake trout larvae was found to contain plasma proteins suggesting an increase in vascular permeability (Guiney et al., 2000
), and the extent of the yolk sac covered by functional blood vessels was reduced in TCDD-exposed rainbow trout sac fry (Hornung et al., 1999
). However, this study relied on the presence of red blood cells to identify blood vessels, and TCDD has well-documented effects on both blood flow (Guiney et al., 2000
; Henry et al., 1997
; Hornung et al., 1999
) and red blood cell number (Belair et al., 2001
), making interpretation of these results difficult. Transgenic zebrafish (Tie2-GFP) have also been employed to examine the effects of TCDD on the vasculature, but this study looked only at the major vessels in the trunk (the caudal artery and vein, intersegmental arteries and veins, the posterior cardinal vein and dorsal aorta) (Belair et al., 2001
). These vessels are maintained throughout the life of the fish with little change and are not affected by TCDD exposure in zebrafish larvae.
The Weinstein lab has developed an atlas of the vasculature in the developing zebrafish. Circulation begins in the zebrafish embryo between 24 and 26 hpf (Isogai et al., 2001). This initial circulation is through a simple circulatory loop in the trunk, which increases in complexity as development progresses. During the course of development specific vessels regress, leading to disconnections and alterations in blood flow (Isogai et al., 2001
).
One vessel identified by Isogai et al. (2001) that appears to undergo extensive remodeling during development is the common cardinal vein (CCV). The CCV, a paired vessel, connects the anterior and posterior cardinal veins on the left and right sides of the fish to the sinus venosus of the heart, providing all venous return to the heart for the first week of life. It is also the only vessel passing over the yolk for the first 60 hpf. Unlike rainbow trout and medaka, the zebrafish never develops a branching network of vitelline vasculature; thus the CCV is the closest equivalent to this vascular structure in zebrafish. Blood flow through the CCV can be detected at the onset of circulation. In dye injection studies, the dye initially spreads out across most of the surface of the yolk (Isogai et al., 2001
). Over the next 4 days the area covered by the dye decreases and moves toward the anterior margin of the yolk sac. By 96 to 120 hpf the dye is fully confined within the CCV and no longer reaches the ventral margin of the yolk sac.
Given the potential importance of the CCV in normal zebrafish development and its relation to vascular structures known to be affected by TCDD in other fish species, the effects of TCDD exposure on development of the CCV in zebrafish were investigated. With the knowledge that loss of the AHR in the mouse results in a failure of specific vessels to regress, it was hypothesized that activation of the AHR by TCDD would accelerate regression of the CCV in developing zebrafish. To test this hypothesis, a detailed description of how the vascular endothelial cells of the CCV develop in control and TCDD-exposed larvae between 27 and 98 hpf was generated. To visualize the CCV, Fli1-eGFP transgenic zebrafish were used where GFP is expressed specifically in the endothelial cells. GFP expression in the CCV is greater in Fli1-eGFP transgenics compared to Tie2-GFP transgenics, and therefore these fish were selected for this study. It was discovered that TCDD significantly reduced CCV area (4462 hpf) and inhibited CCV regression (8096 hpf).
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MATERIALS AND METHODS |
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TCDD exposures.
For the time-course experiments, AB/EK Fli1-eGFP embryos were exposed to TCDD (10 ng/ml) or DMSO (vehicle control) beginning at about 1 hpf in 24-well plates with two embryos per well. After 1 h, the embryos were washed three times and placed in clean 24-well plates. At 24 hpf, the embryos were checked for expression of GFP, and one GFP-expressing embryo per well was kept for all subsequent observations. This experiment was repeated three times for a final n = 30 at each time point. For the dose-response experiment, graded concentrations of TCDD from 0.1 to 10 ng/ml were used. For the zfahr2-MO experiments, 1 ng TCDD/ml was used, because previous studies have shown that high concentrations of TCDD can overcome the protective effect of the morpholino (MO) (Teraoka et al., 2002). All exposures were conducted as described for the time course. Each of these experiments were repeated at least two times for a final n
12. All concentrations of TCDD are nominal, reflecting what was added to the water. Because the plastic of cell culture plates (in cell culture experiments) adsorbs up to
30% of the TCDD added (Hestermann et al., 2000
) the amount of TCDD that actually reached the embryo was less than nominal. Our observations suggest that the concentration of TCDD that reaches the embryo is less when embryos are exposed to the same concentration of waterborne TCDD in plastic compared to exposures in glass.
Morpholino (MO) injections.
Injections were done following the methods of Nasevicius and Ekker (2000) as modified by Prasch et al. (2003)
. The zfahr2-MO sequence was 5'-GTACCGATACCCTCCTACATGGTT-3'. The standard control MO 5'-CTCTTACCTCAGTTACAATTTATA-3' purchased from Gene Tools (Corvallis, OR) was used as an injection control. Embryos were injected at the 1- to 2-cell stage with either the zfahr2-MO or control-MO. The zfahr2-MO was fluorescein tagged, and embryos were screened 12 h after injection. Only successfully injected undamaged embryos were used.
CCV measurements.
Individual larva expressing GFP were dechorionated and placed in 3% methylcellulose prior to being imaged. Zebrafish are able to live and develop normally in this solution for a few days (Westerfield, 1995). Therefore, the larvae were left in the methylcellulose for the duration of the experiment (<48 h) to minimize handling stress. Zebrafish were repeatedly imaged beginning at 27 through 74 hpf (during CCV growth and remodeling) or from 74 through 98 hpf (during CCV regression). Images were taken every 6 h from 32 hpf or 74 hpf. Each larva was positioned on its side so that the connection of the CCV to the anterior and posterior cardinal veins could be seen. Each image was taken at 10x magnification at the same resolution to allow for comparisons between replicates. The CCV of each larva was outlined and the area determined using Scion® Image. All measurements were made "blind" to TCDD treatment and any other variable. Measurements at one time in the initial time course experiment were replicated by a second observer to check for observer bias.
Edema and whole body length measurements.
Pericardial and yolk sac measurements were made by taking DIC images of individual larva at 4x with a constant number of pixels per inch in all images. The perimeter of the pericardial or yolk sac was then outlined and the area determined using Scion® Image. Whole body length was determined by drawing a line from the tip of the snout to the end of the tail using Scion® Image to determine the length in pixels.
Acridine orange.
To assess the number of apoptotic cells, live embryos were soaked in a 10 µg acridine orange/ml (final concentration) in egg water for 15 min in the dark (modified from Abrams et al., 1993; Furutani-Seiki et al., 1996
). The embryos were then removed from this solution, rinsed three times for 5 min each time, and imaged using a fluorescent microscope. The CCV was located by the flow of blood over the yolk. The number of fluorescent cells in the CCV area was counted in each fish (n = 6)
Statistics.
Statistical analysis was done using Statistica (Stat-Soft). For the time course experiment a t-test was employed to compare TCDD to DMSO at each time point independently. For the dose-response studies ANOVA and Tukey's HSD were used to determine significant differences from DMSO controls at each time point independently. No comparisons were made between times. For the MO studies 2-way ANOVA (comparing TCDD treatment and MO treatment) and Tukey's HSD were used. For the MO experiments, no comparisons were made between times. For all experiments p < 0.05 was used to determine significance. A non-parametric test (Mann-Whitney) was used for the regression experiments where the data was not normally distributed.
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RESULTS |
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DISCUSSION |
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CCV Regression
Observations of CCV development in control zebrafish larvae suggest that the decrease in size between 56 and 74 hpf is not due to regression, but rather remodeling. Initially, the CCV develops as a single layer of endothelial cells that spread out across the yolk. Blood cells could clearly be seen escaping out the sides of the CCV, indicating that the early vessel is not a closed tube. By 56 hpf, a double layer of endothelial cells is seen in the most dorsal portion of the CCV. This remodeling of the CCV, from a sheet of endothelial cells to a closed vascular tube, proceeds ventrally down the length of the vessel so that, after 74 hpf, blood flow is confined within the CCV. Thus, it is unlikely that any regression occurred until after 74 hpf.
Starting after 74 hpf, the position of the heart within the pericardium of control larvae changes, and the connection of the heart to the visceral pericardium is lost. Results in control larvae demonstrate that, as the heart migrates dorsally, the CCV also regresses. By 80 hpf, the CCV no longer reaches the ventral edge of the yolk sac. This migration results in a 35% reduction in the length of the CCV in control larvae by 98 hpf. Exposure to 1 or 10 ng TCDD/ml blocks both dorsal movement of the heart and regression of the CCV. A significant difference from controls could be detected at 80 hpf, almost immediately after the onset of migration. The rapid response of this endpoint to TCDD exposure suggests that the effect of TCDD on CCV regression is not secondary to other known endpoints of TCDD toxicity. In TCDD-exposed zebrafish larvae, tube heart formation depends in part on failure of the heart to migrate dorsally. Inhibition of CCV regression by TCDD may contribute to this failure, as dorsal movement of the heart's inflow tract (the CCV) could facilitate dorsal migration of the heart.
Inhibiting translation of AHR2 in the developing zebrafish with zfahr2-MO allows the dependence on AHR2 of TCDD-induced effects on CCV development to be investigated. The zfahr2-MO protected against the TCDD-induced block in CCV regression, demonstrating that this endpoint is dependent on AHR2 expression. Thus, the block in CCV regression joins the list of endpoints of TCDD developmental toxicity in zebrafish that are dependent on AHR2 (Dong et al., 2004; Prasch et al., 2003
; Teraoka et al., 2002
). To date, no endpoint of TCDD developmental toxicity in the zebrafish has been unequivocally found to be independent of AHR2 expression, despite the presence of a second AHR (AHR1) in zebrafish.
Interestingly, knocking down AHR2 expression by itself did not inhibit CCV regression. This is in contrast to the Ahr-null mouse phenotype, where loss of AHR inhibits programmed regression in specific vessels. The presence of AHR1 or incomplete blockage of AHR2 expression by zfahr2- MO could explain this result. The ability of high concentrations of TCDD to overcome the protective effect of the zfahr2-MO suggests that this MO does not fully block AHR2 expression (Teraoka et al., 2002). It is also possible that the mechanism driving programmed regression varies depending on the vessel being examined.
That these results contradicted the initial hypothesis is not entirely unexpected. A number of studies in mice have found that TCDD can both phenocopy the developmental defects seen in Ahr-null mice and produce effects not seen in Ahr-null mice. This suggests a division of AHR-dependent endpoints of TCDD developmental toxicity into two classes. In the first class are those endpoints that are produced by sequestering AHR away from its normal function, and these are also seen in Ahr-null mice. These include immunosuppression (Fernandez-Salguero et al., 1995), dermal hyperplasia with hyperkeratosis (Loertscher et al., 2002
), decreased terminal end buds in the developing mouse mammary gland (Hushka et al., 1998
), and now possibly inhibition of programmed regression in specific vascular beds. In the second class are those endpoints that are seen only after TCDD exposure, suggesting that these result from inappropriate or prolonged activation of the AHR pathway. These include certain changes in gene expression, including increases in xenobiotic metabolizing enzymes (CYP1A), decreases in prostatic buds (Lin et al., 2002
), craniofacial malformations (Courtney, 1976
; Mimura et al., 1997
; Neubert and Dillmann, 1972
), and hydronephrosis (Courtney, 1976
; Lin et al., 2001
). Interestingly, mice expressing a constitutively active AHR have been found to develop stomach tumors (Andersson et al., 2002
), suggesting that the carcinogenic properties of TCDD may be the result of prolonged activation of AHR.
In normal vascular remodeling, a decrease in vascular endothelial growth factor (VEGF) expression accompanied by an increase in angiopoietin 2 (Ang2) expression is involved in stimulating vascular regression (reviewed in Carmeliet, 2000). Results from studies with the Ahr-null mice suggest that activation of AHR by an as yet unknown endogenous ligand (reviewed in Denison and Nagy, 2003
) also plays a role in stimulating regression in specific vessels. The results of this study suggest that TCDD may sequester AHR, preventing it from interacting with an endogenous ligand and thereby blocking regression of the CCV in the developing zebrafish. This could potentially be the result of TCDD-dependent decreased expression of anti-angiogenic factors such as Ang2 or transforming growth factor ß(TGF-ß) or increased expression of factors that lead to stabilization of vessels (i.e., angiopoietin 1). TCDD is known to decrease the expression of TGF-ß in some cell types, including cultured human keratinocytes (Gaido et al., 1992
) and epithelial and mesenchymal cells of the embryonic mouse palate (Abbott and Birnbaum, 1990
). However, a number of studies in fish have reported leakage of serum proteins after TCDD exposures (Dong et al., 2002
; Guiney et al., 2000
), suggesting that an increase in factors that lead to stable vessels, which also tend to decrease leakage from these vessels, is a less probable explanation for the inhibition of regression.
CCV Growth-Remodeling
The failure to detect any apoptotic cells in the CCV, at a time when the difference between TCDD and control CCV area was greatest (56 hpf), coupled with the similar temporal increase in CCV area from 27 to 50 hpf, suggests that TCDD inhibits growth of the CCV early in development, as opposed to accelerating its regression. This inhibition of growth could be the result of decreased proliferation of the endothelial cells or decreased migration of endothelial cells or their precursors to the CCV. A significant reduction in the area covered by the CCV was detected as early as 44 hpf, 4 h earlier than any other sign of TCDD toxicity. Importantly, this is before any changes in yolk sac area resulting from yolk sac edema can be detected. Therefore, differences in yolk sac size are not a factor. Only activation of the AHR pathway, as determined by induction of CYP1A in the vascular endothelium (at 18 hpf, Andreasen et al., 2002b), is seen prior to this endpoint. The next occurring endpoint of TCDD toxicity so far detected is circulation failure in the mesencephalic vein (Dong et al., 2002
). Interestingly, on the rare occasions when circulation was noticeably decreased in the CCV at these early times, the embryos also had extremely small CCVs. This suggests that subtle decreases in blood flow may play a role in the decreased growth of the CCV. Unfortunately, current methods to determine blood flow in zebrafish larvae involve counting individual blood cells as they pass a specific point in a narrow vessel, and the complex and chaotic nature of the blood flow in the CCV precludes such counts being made.
The results of this study suggest that TCDD may inhibit vascular growth of specific vessels in the developing zebrafish. Such a decrease in vascular growth could also explain why Hornung et al. (1999) detected a TCDD-dependent decrease in the number of visible branching points in the vitelline vasculature of rainbow trout. The decrease in CCV area suggesting a decrease in CCV growth could be the result of decreased blood flow, as discussed above, or it could result from changes in expression of angiogenic factors. TCDD has been found to affect the expression of several known angiogenic factors including vascular endothelial growth factor (VEGF) and transforming growth factor (TGF-ß) (Gaido et al., 1992
; Ivnitski-Steele and Walker, 2003
). In the chick embryo, TCDD inhibits coronary vasculogenesis in a VEGF-dependent manner (Ivnitski-Steele and Walker, 2003
). While the reduction in CCV area is an effect on angiogenesis, a reduction in VEGF expression would still result in decreased vascular growth. However, this effect must be localized or limited to a specific VEGF isoform, because the development of other vascular beds appears relatively unaffected (i.e., intersegmental arteries and veins, Belair et al., 2001
). In contrast, a general decrease in VEGF-A expression through the use of a vegf- MO in the developing zebrafish most severely affects the intersegmental arteries and veins (Nasevicius et al., 2000
). A potential explanation for restriction of a VEGF-dependent process to the CCV is a tissue-specific increase in TCDD exposure resulting from the close association of the CCV to the lipid-filled yolk and/or increased exposure to TCDD during yolk resorption.
The results of this study show that TCDD can impair the growth of specific vascular structures and block programmed regression of the vasculature in developing zebrafish. While prior studies have reported a reduction in yolk sac vasculature in TCDD-exposed rainbow trout larvae (Hornung et al., 1999; Spitsbergen et al., 1991
), these studies relied on perfusion of blood to identify vessels. Because TCDD has well-documented effects on blood flow (Guiney et al., 2000
; Henry et al., 1997
; Hornung et al., 1999
) and hematopoiesis (Belair et al., 2001
) in fish larvae, it was not possible to distinguish effects on blood flow or red cell number from effects on vascular endothelial cells in these studies. The results in Ahr-null mice and this study suggest that the loss of normal AHR signaling may affect the development of specific vessels in which, during normal development, vascular regression plays an important role in forming the mature vascular pattern.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI 537052222. Fax: (608) 2653316. E-mail: repeterson{at}pharmacy.wisc.edu.
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