Final Report: Examination of the Estrogen Response Pathways in a New Vertebrate ModelEPA Grant Number: R825297
Title: Examination of the Estrogen Response Pathways in a New Vertebrate Model
Investigators: Linney, Elwood
Institution: Duke University Medical Center
EPA Project Officer: Carleton, James N
Project Period: November 11, 1996 through November 10, 1999
Project Amount: $209,311
RFA: Endocrine Disruptors (1996) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals
Objective:The specific aims to be studied were to: (1) examine the responsiveness of various estrogen response elements (EREs) during these same stages of development using both transient DNA injections of DNA constructs having EREs coupled to the a simple thymidine kinase promoter driving the green fluorescent protein (GFP), or through the production of transgenic fish having these genes incorporated, as new genetic material, into their genome; and (2) examine both the appearance of estrogen mRNA during development and the appearance of estrogen receptor responsiveness after the administration of various estrogen-like chemicals. This report will be directed towards results involving these specific aims.
During the research period, we were able to develop the following technologies that are relevant to the specific aims: (1) production of expressing transgenic zebrafish both via DNA microinjection and pseudotyped retroviral vector infection; (2) development of estrogen response transgenic zebrafish whose expression could be monitored by fluorescence capture of reporter gene activity; and (3) isolation, characterization, and study of a zebrafish estrogen receptor beta.
At the onset of this work, there was some question concerning whether transgenic zebrafish could be made that expressed reporter gene activity from developmentally regulated promoters. In our first publication, we described how we worked out conditions to routinely obtain transgene expression in zebrafish. In addition, there had been a serious question regarding whether a non-enzymatic reporter gene such as the fluorescent reporter, Green Fluorescent Protein (GFP), could provide enough of a signal to be routinely used to detect gene activity. We found, from our experience, that this could be done with careful consideration of the detection equipment used to detect the fluorescent gene. We had to modify our procedures somewhat to examine different mutant forms of the GFP gene. The zebrafish embryo can produce considerable autofluorescence that can obscure the reporter fluorescence. In our studies with different color derivatives of GFP plus a fluorescent enzymatic reporter (beta-lactamase with fluorescent substrate), we found the following qualitative results:
worse best BFP, lactamase>CFP>eGFP>YFP
The YFP reporter was best because it minimized autofluorescence from the embryo and still was a bright reporter gene product.
Other modifications that we had to make included transferring our transgenic lines into zebrafish lines that minimized natural pigmentation. Embryonic pigmentation appears within 24 hours and this causes fluorescence quenching of the reporter signal. In these studies, we chose to breed the transgenic lines we produced into a homozygous albino background. This has allowed us to follow fluorescence in individual cells in the embryo and larvae for as long as 18 days (or certainly beyond the 3-day embryonic period).
Some of this work took considerable time organizing our equipment and identifying how we could probably capture fluroescent images of developing zebrafish. At the present time, we can embed live embryos in agarose, mount them on a temperature-controlled coverslip apparatus, and then write computer scripts so that our computer will take defined fluorescent image captures every 10-30 minutes. We have been able to capture time-lapse studies of developing zebrafish for over 2 days. These images can then be converted to quick-time movies for demonstration of developmental gene expression on computers or computer projectors. While there are several minor problems that from time to time occur during image capture, this type of analysis allows for visualization of gene expression during embryonic development and the viewing of embryonic events, which cannot be done with mammalian embryonic models at this time.
Zebrafish ER-beta isolation. Before I go into the work specifically involving estrogen response element driven transgenic fish, I will describe the work done in isolating a zebrafish estrogen receptor. Using consensus oligonucleotides for regions shared by estrogen receptors from several different species, we used RNA from adult female liver and ovaries as a target for RT-PCR amplification of estrogen receptor sequences. Cloning and sequencing of our product confirmed that at least one clone was estrogen receptor-like. We then used this partial cDNA to isolate a full-length estrogen receptor cDNA from a cDNA library from 30 day old zebrafish.
The receptor we isolated appears to be an estrogen receptor beta. It is very homologous to the published goldfish ER-beta. Transfection experiments with a zebrafish liver cell line and firefly luciferase reporter genes show that it is an estradiol inducible receptor. Using RNAse-resistant RNA-RNA hybridization analysis and acrylamide gel electrophoresis, we showed that the concentration of this mRNA is very low during embryogenesis and increases into the larval and adult stages. An antisera has been produced against the carboxyl end region of the receptor and this antisera allows us to identify ER-beta using Western blotting procedures. We have not yet examined whether this antisera could be used to in situ localize the receptor.
We have isolated genomic sequences from the 5' end of the gene and appear to have at least some of the promoter region cloned for regulation analysis. Our plans are to try to identify the complete promoter region so that it could be used to derive transgenic reporter fish displaying fluorescence in those regions and at that developmental time when the ER-beta gene is expressed. It also should help us to identify the DNA controlling elements that mediate its expression.
Zebrafish estrogen response element (ERE) driven transgenic lines. We have made transgenic zebrafish lines with an estrogen response element coupled to the Herpes simplex virus TK promoter. In zebrafish liver cell lines, when we transfect this regulatory sequence coupled to a firefly luciferase reporter gene, we can get estradiol induction of luciferase. In our transgenic lines, we have coupled this regulatory sequence to a green fluorescent protein (GFP) gene modified to target the protein to the nucleus. We have isolated and partially characterized two transgenic lines. The expression of GFP appears in a limited number of cells (20-50) detectable 24 hours postfertilization. The cells appear to be migrating along the pronephric ducts and over the course of 14 days, the GFP+ cells migrate to and aggregate around the pronephros. At this point in time, we have not clearly anatomically identified this site of aggregation/association. To perform these studies, we had to breed one of our transgenic lines into an albino/albino background. Time-lapse microscopy has been performed to create quick-time movies of this migration. The first 2 days of detectable GFP expression in this limited number of cells suggest that these migrating cells might be precursors to primordial germ cells. However, evidence from other laboratories suggest that primordial germ cells migrate ventral to the pronephric ducts at day 2 or 3, while the GFP+ cells migrate along the pronephric ducts. We have not ruled out the possibility that the early GFP+ cells are precursors to the primordial germ cells and with loss of GFP expression, migrate ventrally to the primitive gonad. We have generated antisera to the zebrafish ER-beta and to the zebrafish vasa protein (a germ cell marker), but we do not as yet know whether these antisera could be used in situ to localize ER+ cells and vasa+ cells.
Our preliminary attempts at treating these cells lines with estradiol or anti-estrogens have revealed no distinct change in the GFP fluorescence. We are beginning to train ourselves on a laser ablator so that we might eliminate the small number of cells that are expressing GFP to determine how this might affect the phenotype of the organism. It is difficult to measure quantitative differences of fluorescence in living embryos, particularly because we have a saturating amount of reporter protein in cells; therefore, we are looking for other procedures and reagents for determining the estrogen responsiveness of these transgenic fish. Preliminary experiments with administration of estradiol to adult females result in an increase if GFP in regions of the reproductive tract. We hope to use quantitation PCR analysis to investigate a change of reporter RNA at an individual embryo level.