A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Xenopus Tadpoles

Increasing numbers of substances present in the environment are postulated to have endocrine-disrupting effects on vertebrate populations. However, data on disruption of thyroid signaling are fragmentary, particularly at the molecular level. Thyroid hormone (TH; triiodothyronine, T(3)) acts principally by modulating transcription from target genes; thus, thyroid signaling is particularly amenable to analysis with a transcriptional assay. Also, T(3) orchestrates amphibian metamorphosis, thereby providing an exceptional model for identifying thyroid-disrupting chemicals. We combined these two advantages to develop a method for following and quantifying the transcriptional action of T(3) in Xenopus laevis tadpoles. This technology provides a means of assessing thyroid activity at the molecular level in a physiologically relevant situation. Moreover, translucent tadpoles are amenable to “on-line” imaging with fluorescent reporter constructs that facilitate in vivo measurement of transcriptional activity. We adapted transgenesis with TH-responsive elements coupled to either luciferase or green fluorescent protein to follow T(3)-dependent transcription in vivo. To reduce time of exposure and to synchronize responses, we optimized a physiologic pre-treatment protocol that induced competence to respond to T(3) and thus to assess T(3) effects and T(3) disruption within 48 hr. This pretreatment protocol was based on a short (24 hr), weak (10(−12) M) pulse of T(3) that induced TH receptors, facilitating and synchronizing the transcriptional responses. This protocol was successfully applied to somatic and germinal transgenesis with both reporter systems. Finally, we show that the transcriptional assay allows detection of the thyroid-disrupting activity of environmentally relevant concentrations (10(−8) M) of acetochlor, a persistent herbicide

Assessment of Estrogenic Endocrine-Disrupting Chemical Actions in the Brain Using in Vivo Somatic Gene Transfer

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element–thymidine kinase–luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17β-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 μg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo

Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors

BACKGROUND: PAX6 is a transcription factor playing a crucial role in the development of the eye and in the differentiation of the pancreatic endocrine cells as well as of enteroendocrine cells. Studies on the mouse Pax6 gene have shown that sequences upstream from the P0 promoter are required for expression in the lens and the pancreas; but there remain discrepancies regarding the precise location of the pancreatic regulatory elements. RESULTS: Due to genome duplication in the evolution of ray-finned fishes, zebrafish has two pax6 genes, pax6a and pax6b. While both zebrafish pax6 genes are expressed in the developing eye and nervous system, only pax6b is expressed in the endocrine cells of the pancreas. To investigate the cause of this differential expression, we used a combination of in silico, in vivo and in vitro approaches. We show that the pax6b P0 promoter targets expression to endocrine pancreatic cells and also to enteroendocrine cells, retinal neurons and the telencephalon of transgenic zebrafish. Deletion analyses indicate that strong pancreatic expression of the pax6b gene relies on the combined action of two conserved regulatory enhancers, called regions A and C. By means of gel shift assays, we detected binding of the homeoproteins PDX1, PBX and PREP to several cis-elements of these regions. In constrast, regions A and C of the zebrafish pax6a gene are not active in the pancreas, this difference being attributable to sequence divergences within two cis-elements binding the pancreatic homeoprotein PDX1. CONCLUSION: Our data indicate a conserved role of enhancers A and C in the pancreatic expression of pax6b and emphasize the importance of the homeoproteins PBX and PREP cooperating with PDX1, in activating pax6b expression in endocrine pancreatic cells. This study also provides a striking example of how adaptative evolution of gene regulatory sequences upon gene duplication progressively leads to subfunctionalization of the paralogous gene pair

Expression of the microphthalmia-associated basic helix-loop-helix leucine zipper transcription factor Mi in avian neuroretina cells induces a pigmented phenotype

The microphthalmia gene (mi) appears to be required for pigment cell development, based on its mutation in mi mice. The mi gene encodes a basic helix-loop-helix leucine zipper transcription factor (Mi) with tissuerestricted expression. To investigate the role of mi in cell proliferation and pigmentation, we transfected neuroretina (NR) cells with a recombinant virus expressing the murine mi cDNA. The virus induced the proliferation of chicken NR cells in response to fibroblast growth factor 2, which enabled them to form colonies in soft agar. In contrast to control cultures, transfected chicken NR cells or quail NR cells became rapidly pigmented and strongly expressed the QNR-71 mRNA encoding a melanosomal protein. These results demonstrate that Mi not only acts as pigmentation inducer but is also able to modulate the response of cells to growth factors.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Tadpoles-0

<p><b>Copyright information:</b></p><p>Taken from "A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Tadpoles"</p><p>Environmental Health Perspectives 2005;113(11):1588-1593.</p><p>Published online 11 Jul 2005</p><p>PMCID:PMC1310923.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Tadpoles-5

<p><b>Copyright information:</b></p><p>Taken from "A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Tadpoles"</p><p>Environmental Health Perspectives 2005;113(11):1588-1593.</p><p>Published online 11 Jul 2005</p><p>PMCID:PMC1310923.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p

Met909 plays a key role in the activation of the progesterone receptor and also in the high potency of 13-ethyl progestins

ABSTRACT Many progestins have been developed for use in contraception, menopausal hormone therapy, and treatment of gynecological diseases. They are derived from either progesterone or testosterone, and they act by binding to the progesterone receptor (PR), a hormone-inducible transcription factor belonging to the nuclear receptor superfamily. Unlike mineralocorticoid, glucocorticoid, and androgen receptors, the steroid-receptor contacts that trigger the switch of the ligand-binding domain from an inactive to an active conformation have not yet been identified for the PR. With this aim, we solved the crystal structure of the ligand-binding domain of the human PR complexed with levonorgestrel, a potent testosterone-derived progestin characterized by a 13-ethyl substituent. Via mutagenesis analysis and functional studies, we identified Met909 of the helix 12 as the key residue for PR activation by both testosterone-and progesterone-derived progestins with a 13-methyl or a 13-ethyl substituent. We also showed that Asn719 contributes to PR activation by testosterone-derived progestins only, and that Met759 and Met909 are responsible for the high potency of 19-norprogestins and of 13-ethyl progestins, respectively. Our findings provide a structural guideline for the rational synthesis of potent PR agonist and antagonist ligands that could have therapeutic uses in women&apos;s health

A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Tadpoles-2

<p><b>Copyright information:</b></p><p>Taken from "A Rapid, Physiologic Protocol for Testing Transcriptional Effects of Thyroid-Disrupting Agents in Premetamorphic Tadpoles"</p><p>Environmental Health Perspectives 2005;113(11):1588-1593.</p><p>Published online 11 Jul 2005</p><p>PMCID:PMC1310923.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.</p