Warren Heideman

Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.

Aryl Hydrocarbon Receptor Activation Produces Heart-Specific Transcriptional and Toxic Responses in Developing Zebrafish

Proper regulation of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, is required for normal vertebrate cardiovascular development. AHR hyperactivation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during zebrafish (Danio rerio) development results in altered heart morphology and function, culminating in death. To identify genes that may cause cardiac toxicity, we analyzed the transcriptional response to TCDD in zebrafish hearts. Zebrafish larvae were exposed to TCDD for 1 h at 72 h after fertilization (hpf), and the hearts were extracted for microarray analysis at 1, 2, 4, and 12 h after exposure (73, 74, 76, and 84 h postfertilization). The remaining body tissue was also collected at each time for comparison. TCDD rapidly induced expression in 42 genes within 1 to2hof exposure. These genes function in xenobiotic metabolism, proliferation, heart contractility, and pathways that regulate heart development. Furthermore, these expression changes preceded signs of cardiovascular toxicity, characterized by decreased stroke volume, peripheral blood flow, and a halt in heart growth. This identifies strong candidates for important AHR target genes. It is noteworthy that the TCDD-induced transcriptional response in the hearts of zebrafish larvae was substantially different from that induced in the rest of the body tissues. One of the biggest differences included a cluster of genes that were down-regulated 12 h after exposure in heart tissue, but not in the body samples. More than 70% of the transcripts in this heart-specific cluster promote cellular growth and proliferation. Thus, the developing heart stands out as being responsive to TCDD at both the level of toxicity and gene expression.

Latent TGF-β binding protein 3 identifies a second heart field in zebrafish.

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary outflow tract but not a right ventricle, the existence and function of SHF-like cells in these species has remained a topic of speculation. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss-of-function studies in zebrafish, a lower vertebrate with a single ventricle, that latent TGF-β binding protein 3 (ltbp3) transcripts mark a field of cardiac progenitor cells with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3+ cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the outflow tract and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGF-β ligands, zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of cardiac progenitor cells in both fields. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3+ cells due to compromised progenitor proliferation. Furthermore, small-molecule inhibition of TGF-β signalling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGF-β type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3–TGF-β signalling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Regulation of the Cln3–Cdc28 kinase by cAMP in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae grows at widely varying rates in different growth media. In order to maintain a relatively constant cell size, yeast cells must regulate the rate of progress through the cell cycle to match changes in growth rate, moving quickly through G1 in rich medium, and slowly in poor medium. We have examined connections between nutrients, and the expression and activity of Cln3–Cdc28 kinase that regulates the G1–S boundary of the cell cycle in yeast, a point referred to as Start. We find that Cln3 protein levels are highest in glucose and lower in poorer carbon sources. This regulation involves both transcriptional and post‐transcriptional control. Although the Ras–cAMP pathway does not appear to affect CLN3 transcription, cAMP increases Cln3 protein levels and Cln3–Cdc28 kinase activity. This regulation requires untranslated regions of the CLN3 message, and can be explained by changes in protein synthesis rates caused by cAMP. A model for CLN3 regulation and function is presented in which CLN3 regulates G1 length in response to nutrients.

Disruption of erythropoiesis by dioxin in the zebrafish

2,3,7,8‐Tetrachlorodibenzo‐p‐ dioxin (TCDD, or dioxin) causes early life stage mortality in a variety of fish species. We have used the zebrafish (Danio rerio) to study the cardiovascular effects of TCDD treatment over the time course of zebrafish development. Early TCDD exposure (6 ng/ml) starting at 4 hr postfertilization (hpf) produced reductions in blood flow and in the number of circulating erythrocytes. These defects were consistently observable by 72 hpf. However, these responses were not observed when TCDD exposure was delayed until 96 hpf or later. These results suggest a model in which TCDD interferes with cardiovascular and erythropoietic developmental processes that are normally completed by 96 hpf. This model is strengthened by the finding that TCDD exposure blocks the step in hematopoiesis in which developing zebrafish switch from the primitive phase to the definitive phase of erythropoiesis. We observed no effect of TCDD on the levels of circulating primitive erythrocytes before 72 hpf and the expression of markers for early hematopoiesis, GATA‐1 and GATA‐2. However, early TCDD exposure prevented the appearance of definitive phase erythrocytes. TCDD produced a small delay in the migration of blood cells expressing SCL from the intermediate cell mass to the dorsal mesentery and dorsal aorta. Despite the decrease in blood flow produced by TCDD, confocal microscopy of the trunk vasculature by using a Tie2/green fluorescence protein endothelial marker at 48, 60, 72, and 96 hpf of TCDD‐exposed (4 hpf) revealed no apparent defects in blood vessel structure.

Titanium dioxide nanoparticles produce phototoxicity in the developing zebrafish

Abstract Exposure of humans and other organisms to nanomaterials is increasing exponentially. It is important, but difficult, to predict the biological consequences of these exposures. We hypothesized that the unique chemical properties that make nanoparticles useful might also be the key in predicting their biological impact. To investigate this, we chose titanium dioxide nanoparticles (TiO2NPs) and developing zebrafish embryos as model systems. TiO2NPs absorb photons to generate electron–hole pairs that react with water and oxygen to form cytotoxic reactive oxygen species (ROS). Here, we show that the exposure of zebrafish embryos to TiO2NPs produces malformation and death, but only if the fish are also illuminated. TiO2NPs are taken up into the developing fish, but the egg chorion is a barrier to uptake until the embryos hatch. Chemical probes and a transgenic reporter line confirm photo-dependent production of ROS in vivo, and the addition of an ROS scavenger rescues fish embryos from toxicity. To our knowledge, this is the first study to show a photo-dependent toxic response in a whole organism from exposure to TiO2NPs. Of further significance, our study highlights the relationship between the property of the material that makes it useful and the biological effect that is produced. This concept should serve as a guide for future nanotoxicological studies aiming to identify potential hazardous effects on organisms.

Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions.

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.

Influence of Humic Acid on Titanium Dioxide Nanoparticle Toxicity to Developing Zebrafish

Titanium dioxide nanoparticle (TiO2NP) suspension stability can be altered by adsorption of dissolved organic matter (DOM). This is expected to impact their environmental fate and bioavailability. To date, the influence of DOM on the toxicity of TiO2NPs to aquatic vertebrates has not been reported. We examined the impact of Suwannee River humic acid (HA) on the toxicity of TiO2NPs to developing zebrafish (Danio rerio) in the dark and under simulated sunlight illumination. Adsorption of HA increased suspension stability and decreased TiO2NP exposure. TiO2NPs were more toxic in the presence of HA. In the absence of simulated sunlight, a small but significant increase in lethality was observed in fish exposed to TiO2NPs in the presence of HA. Under simulated sunlight illumination, photocatalytic degradation of HA reduced suspension stability. Despite the lower concentrations of Ti associated with fish in the treatments containing HA, under simulated sunlight illumination, median lethal concentrations were lower and oxidative DNA damage was elevated relative to fish exposed to TiO2NPs in the absence of HA. This study demonstrates the importance of considering environmental factors (i.e., exposure to sunlight, adsorption of DOM) when assessing the potential risks posed by engineered nanomaterials in the environment.

Reproductive and Developmental Toxicity of Dioxin in Fish

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a global environmental contaminant and the prototypical ligand for investigating aryl hydrocarbon receptor (AHR)-mediated toxicity. Environmental exposure to TCDD results in developmental and reproductive toxicity in fish, birds and mammals. To resolve the ecotoxicological relevance and human health risks posed by exposure to dioxin-like AHR agonists, a vertebrate model is needed that allows for toxicity studies at various levels of biological organization, assesses adverse reproductive and developmental effects and establishes appropriate integrative correlations between different levels of effects. Here we describe the reproductive and developmental toxicity of TCDD in feral fish species and summarize how using the zebrafish model to investigate TCDD toxicity has enabled us to characterize the AHR signaling in fish and to better understand how dioxin-like chemicals induce toxicity. We propose that such studies can be used to predict the risks that AHR ligands pose to feral fish populations and provide a platform for integrating risk assessments for both ecologically relevant organisms and humans.

Glucose Regulation of Saccharomyces cerevisiae Cell Cycle Genes

ABSTRACT Nutrient-limited Saccharomyces cerevisiae cells rapidly resume proliferative growth when transferred into glucose medium. This is preceded by a rapid increase in CLN3, BCK2, and CDC28 mRNAs encoding cell cycle regulatory proteins that promote progress through Start. We have tested the ability of mutations in known glucose signaling pathways to block glucose induction of CLN3, BCK2, and CDC28. We find that loss of the Snf3 and Rgt2 glucose sensors does not block glucose induction, nor does deletion of HXK2, encoding the hexokinase isoenzyme involved in glucose repression signaling. Rapamycin blockade of the Tor nutrient sensing pathway does not block the glucose response. Addition of 2-deoxy glucose to the medium will not substitute for glucose. These results indicate that glucose metabolism generates the signal required for induction of CLN3, BCK2, and CDC28. In support of this conclusion, we find that addition of iodoacetate, an inhibitor of the glyceraldehyde-3-phosphate dehydrogenase step in yeast glycolysis, strongly downregulates the levels CLN3, BCK2, and CDC28 mRNAs. Furthermore, mutations in PFK1 and PFK2, which encode phosphofructokinase isoforms, inhibit glucose induction of CLN3, BCK2, and CDC28. These results indicate a link between the rate of glycolysis and the expression of genes that are critical for passage through G1.