Fipronil Affects Craniofacial and Heart Development in Zebrafish Embryos (Danio rerio).

Fipronil is a broad-spectrum insecticide that has off-target effects in developing vertebrate embryos. In this study, we investigate treatment of zebrafish embryos with fipronil over the course of 5 days and examine the effects on body length, the cardiovascular system, and craniofacial morphology. We found the insecticide caused shorter body length and a decrease in eye size. By examining specific heart chamber morphology, as well as jaw angle and length, we quantified defects including enlargement of the heart and increases in jaw length and width. Further studies are needed to assess the mechanisms of fipronil's effect on vertebrate development for both environmental and human health concerns.

Spironolactone affects cardiovascular and craniofacial development in zebrafish embryos (Danio rerio).

Spironolactone, a potassium-sparing diuretic and aldosterone antagonist, is a mineralocorticoid hormone commonly prescribed to patients suffering from heart failure, hirsutism, dermatological afflictions, and hypertension. Interestingly, relatively little work has been done on the development of vertebrate embryos after exposure to this compound. Here, we treat zebrafish embryos with spironolactone at 10-6 M, 10-7 M, or 10-8 M, and observe them after three to seven days of exposure. While no effect was observed in mortality, we did detect differences in cardiovascular development at 3 dpf and craniofacial development at 5 dpf. At 10-6 M, smaller atria, ventricles, and blood vessels were observed. The highest concentrations also caused a longer ceratohyal/Meckel's distance, longer palatoquadrate, and smaller angles between the palatoquadrate and both the ceratohyal and Meckel's. Further research of spironolactone's effects on embryonic development could lead to a better understanding of the compound resulting in improved public and environmental health.

Atrazine affects craniofacial chondrogenesis and axial skeleton mineralization in zebrafish ( Danio rerio).

Atrazine is a commonly used herbicide that has previously been implicated as an endocrine-disrupting compound. Previous studies have shown that estrogenic endocrine-disrupting compounds affect the development of the heart, cartilage, and bone in zebrafish ( Danio rerio). To determine whether atrazine has effects similar to other endocrine disruptors, zebrafish embryos were treated with a range of atrazine concentrations. Atrazine treatment at a low concentration of 0.1 µM resulted in significant differences in craniofacial cartilage elements, while concentrations ≥1 µM led to decreased survival and increased heart rates. Fish treated with ≥1 µM atrazine also developed with delayed vertebrae mineralization. Higher concentrations of atrazine caused gross craniofacial defects and decreased hatching rates. Further studies into the molecular pathways disrupted in these developmental processes could shed light on a link between endocrine-disrupting compounds and developmental abnormalities.

Nuclear Androgen Receptor Regulates Testes Organization and Oocyte Maturation in Zebrafish.

Androgens act through the nuclear androgen receptor (AR) to regulate gonad differentiation and development. In mice, AR is necessary for spermatogenesis, testis development, and formation of external genitalia in males and oocyte maturation in females. However, the extent to which these phenotypes are conserved in nonmammalian vertebrates is not well understood. Here, we generate zebrafish with a mutation in the ar gene (aruab105/105) and examine the role of AR in sexual determination and gonad development. We found that zebrafish AR regulates male sexual determination, because the majority of aruab105/105 mutant embryos developed ovaries and displayed female secondary sexual characteristics. The small percentage of mutants that developed testes displayed female secondary sexual characteristics, exhibited structurally disorganized testes, and were unable to release or produce normal levels of sperm, demonstrating that AR is necessary for zebrafish testis development and fertility. In females, we found that AR regulates oocyte maturation and fecundity. The aruab105/105 mutant females developed ovaries filled primarily with immature stage I oocytes and few mature stage III oocytes. Two genes whose expression is enriched in wild-type ovaries compared with testes (cyp19a1a, foxl2a) were upregulated in ar mutant testes, and two genes enriched in testes (amh, dmrt1) were upregulated in ar mutant ovaries. These findings demonstrate that AR regulates sexual determination, testis development, and oocyte maturation and suggest that AR regulates sexually dimorphic gene expression. The ar mutant we developed will be useful for modeling human endocrine function in zebrafish.

Investigation of the effects of estrogen on skeletal gene expression during zebrafish larval head development.

The development of craniofacial skeletal structures requires well-orchestrated tissue interactions controlled by distinct molecular signals. Disruptions in normal function of these molecular signals have been associated with a wide range of craniofacial malformations. A pathway mediated by estrogens is one of those molecular signals that plays role in formation of bone and cartilage including craniofacial skeletogenesis. Studies in zebrafish have shown that while higher concentrations of 17-ß estradiol (E 2) cause severe craniofacial defects, treatment with lower concentrations result in subtle changes in head morphology characterized with shorter snouts and flatter faces. The molecular basis for these morphological changes, particularly the subtle skeletal effects mediated by lower E 2 concentrations, remains unexplored. In the present study we address these effects at a molecular level by quantitative expression analysis of sets of candidate genes in developing heads of zebrafish larvae treated with two different E 2 concentrations. To this end, we first validated three suitable reference genes, ppia2, rpl8 and tbp, to permit sensitive quantitative real-time PCR analysis. Next, we profiled the expression of 28 skeletogenesis-associated genes that potentially respond to estrogen signals and play role in craniofacial development. We found E 2 mediated differential expression of genes involved in extracellular matrix (ECM) remodelling, mmp2/9/13, sparc and timp2a, as well as components of skeletogenic pathways, bmp2a, erf, ptch1/2, rankl, rarab and sfrp1a. Furthermore, we identified a co-expressed network of genes, including cpn1, dnajc3, esr1, lman1, rrbp1a, ssr1 and tram1 with a stronger inductive response to a lower dose of E 2 during larval head development.

Bis-GMA affects craniofacial development in zebrafish embryos (Danio rerio).

Estrogen is a steroid hormone that is vital in vertebrate development and plays a role in a variety of developmental processes including cartilage and craniofacial formation. The effects of estrogen can be mimicked by other compounds found in the environment known as xenoestrogens. Bisphenol-A (BPA) is a known xenoestrogen and is combined with glycidyl methacrylate to make Bisphenol A glycidyl methacrylate (Bis-GMA), a major component in dental resin based composites (RBCs). Bis-GMA based RBCs can release their components into the saliva and bloodstream. Exposure to 1µM and 10µM Bis-GMA in Danio rerio embryos results in increased mortality of approximately 30% and 45% respectively. Changes to gross morphology, specifically craniofacial abnormalities, were seen at concentrations as low as 10nM. While the molecular pathways of Bis-GMA effects have not been studied extensively, more is known about one of the components, BPA. Further research of Bis-GMA could lead to a better understanding of xenoestrogenic activity resulting in improved public and environmental health.

Modulation of estrogen causes disruption of craniofacial chondrogenesis in Danio rerio.

Estrogen is a steroid hormone that is ubiquitous in vertebrates, but its role in cartilage formation has not been extensively studied. Abnormalities of craniofacial cartilage and bone account for a large portion of birth defects in the United States. Zebrafish (Danio rerio) have been used as models of human disease, and their transparency in the embryonic period affords additional advantages in studying craniofacial development. In this study, zebrafish embryos were treated with 17-ß estradiol (E2) or with an aromatase inhibitor and observed for defects in craniofacial cartilage. Concentrations of E2 greater than 2µM caused major disruptions in cartilage formation. Concentrations below 2µM caused subtle changed in cartilage morphology that were only revealed by measurement. The angles formed by cartilage elements in fish treated with 1.5 and 2µM E2 were increasingly wide, while the length of the primary anterior-posterior cartilage element in these fish decreased significantly from controls. These treatments resulted in fish with shorter, flatter faces as estrogen concentration increased. Inhibition of aromatase activity also resulted in similar craniofacial disruption indicating that careful control of estrogen signaling is required for appropriate development. Further investigation of the phenomena described in this study could lead to a better understanding of the etiology of craniofacial birth defects and endocrine disruption of cartilage formation.

Estrogen prevents cardiac and vascular failure in the 'listless' zebrafish (Danio rerio) developmental model.

The presence of a robust estrogen (E2) response system throughout heart and blood vessel tissues of vertebrates, including humans, has led to the speculation that this ubiquitous hormone may play a prominent role in the health and maintenance of the adult cardiovascular system (CVS). We previously established an embryonic zebrafish model called 'listless', which results from the inhibition of E2 synthesis by treatment with aromatase enzyme inhibitors (AI). These fish have outward symptoms similar to the human condition of congestive heart failure and tamponade. E2 replacement therapy (1) reduced the severity of cardiac sac abnormalities, (2) protected heart function, (3) prevented reduction in heart size, and (4) reduced blood vessel deterioration. Nitric oxide may be a critical downstream mediator of these events. We also demonstrate that removal of fluid around the heart increases survival of AI-treated fish. These results strongly indicate the importance of E2 in the developing CVS of the zebrafish and offer a potential model for the study of its role in CVS development, maintenance, and disease conditions.

Zebrafish (Danio rerio) androgen receptor: sequence homology and up-regulation by the fungicide vinclozolin.

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.

Embryonic expression and steroid regulation of brain aromatase cyp19a1b in zebrafish (Danio rerio).

Estradiol is produced from testosterone by the aromatase gene, cyp19. In the zebrafish Danio rerio, brain aromatase, cyp19a1b, is highly expressed during development. We report the developmental expression pattern of cyp19a1b using whole mount in situ hybridization and describe hormonal effects on the gene using RT-PCR. Expression is up-regulated between 24 and 48 hours postfertilization (hpf). Localized expression of cyp19a1b is first detected at 48 hpf in the preoptic area, hypothalamus, terminal nerve, and olfactory bulb. The gene is itself induced by estradiol in a positive feedback loop. Testosterone exposure also induces the cyp19a1b gene in zebrafish; however, a majority of this induction is blocked by an estrogen receptor antagonist. The expression pattern of aromatase in the brain and its control by steroid hormones is well conserved among the vertebrate lineage.

Genomic structure and embryonic expression of estrogen receptor beta a (ERbetaa) in zebrafish (Danio rerio).

Estrogenic steroid hormones mediate complex actions important in both embryonic and adult life. The hormones signal through ligand-inducible transcription factors known as estrogen receptors (ERs). In this study, we have isolated a zebrafish estrogen receptor with homology to human estrogen receptor beta (ERbeta). This zebrafish ERbeta (ERbetaa) has a conserved genomic structure of eight coding exons with boundaries similar to those of human ERbeta. The coding exon structures of two other zebrafish estrogen receptors (ERalpha and ERbetab) are presented as well. We also analyzed 3.3 kb of the promoter region and identified numerous putative transcription factor binding sites, including SP1 and ER half sites. Zebrafish ERbetaa message RNA is maternally loaded, but quickly degraded after fertilization, as detected by reverse transcriptase polymerase chain reaction. ERbetaa transcripts are detected again between 24 and 48 h post fertilization. These results indicate that ERbeta has been highly conserved during evolution and is likely used during later embryogenesis in zebrafish. Future identification of the expression levels and patterns of this and other estrogen receptors in zebrafish will allow a better understanding of estrogen signaling during embryogenesis.