The research program in this laboratory has moved through different areas of interest, most of them involved with how embryonic cells develop. Our early work with embryonal carcinoma cells (EC cells, the conceptual precursor to embryonic stem cells) involved using viruses to determine the transcriptional control differences between stem and differentiated cell. We found the restriction to expression and replication of polyoma virus in EC cells could be overcome by isolated enhancer mutants in polyoma (Fujimura et al, 1981, J.Virol.; Fujimura et al. 1981, Cell; Fujimura et al. 1982, PNAS)--these could have changes as small as a base pair change though a stronger effect involved duplications around these single base changes (Linney and Donerly, 1983, Cell)--these sequences were used by Mario Capecchi in his first expression constructs in embryonic stem cells and we used them to study the embryonic need for enhancers in mouse embryos (Martinez-Salas et al. 1989, Genes Dev.). We then explored the restriction to retroviral gene expression in stem cells and found both an enhancer restriction plus an apparent instability of the 5' end of retroviral mRNAs in stem cells (Linney et al. 1984, Nature; Davis et al, 1985, Nature) . We made modifications in retroviral vectors to allow for expression in stem cells (Linney et al. 1987, J. Virol.)
The EC cells were working with would differentiate when exposed to retinoic acid. When the retinoic acid receptors were discovered by other laboratories (members of the steroid superfamily of receptors) we made dominant negative vectors of these and showed that they would inhibit EC cells from differentiation due to retinoic acid exposure, implicating the retinoic acid receptors in this differentiation event (Espeseth et al, 1989, Genes Dev.).
I was then asked by the Cancer Center to setup and direct a Shared Transgenic Mouse Facility for cancer center members. Several cancer center members used this and our own work with transgenics was centered on examining retinoic acid receptor signaling in mouse embryos. We developed indicator transgenics that displayed, through a beta-galactosidase reporter, a subset of regions of natural retinoic acid receptor activity. These lines were also inducible to ectopic induction of the transgene through gavage feeding of pregnant females with retinoic acid (Balkan et al. 1992, PNAS). We also developed transgenic lines that had directed to specific tissue, constitutively active retinoic acid receptors coupled to beta-galactosidase to produce teratogenic phenotypes in the tissue that resembled the teratogenic effects of exogenous retinoic acid exposure (Balkan et al. 1992, Dev. Biol; Underhill et al. 1994, J. Cell Biol.).
After the transgenic facility was working smoothly I stepped down to concentrate on my own laboratory's research. During that time I spent a sabbatical year on campus in the Center for In vivo Microscopy developing a 3 dimensional atlas of mouse embryonic development using their magnetic resonance microscopy technology (Smith et al. 1994, PNAS). This work led me to another transition, that of switching from cell culture and mouse embryos to zebrafish because the 3D imaging of fixed stages of mouse embryos attracted me to the idea of studying an embryo that developed outside the mother, and an embryo that remained sufficiently small in size so that we could make transgenic lines with fluorescent reporters to record real-time expression of genes during embryonic development.
We, and others, have successfully done this. We were the first to make expressing transgenic lines of zebrafish by infecting embryos with pseudotyped retroviral vectors (Linney et al. 1999, Dev. Biol.). We were the first to make inducible transgenic lines of zebrafish using sequences similar to those we used in making retinoic acid responsive transgenic mice (Perz-Edwards et al. 2001, Dev. Biol.). We have both reviewed the field of zebrafish transgenics (Udvadia and Linney, 2003, Dev. Biol.) and published a chapter on our techniques (Linney and Udvadia, 2004, Methods in Mol. Biol.).
At that point in time, our research with zebrafish split into two distinct areas and remains split into these divisions: 1) environmentally, using zebrafish to study the sensitivity of the developing nervous system to compounds that effect neuronal signaling (Linney et al. 2004, Neurotox. Teratol.; Roy and Linney, 2008, Source book of models for biomedical research)--i.e. pesticides that impact upon acetylcholine, GABA and glycine receptors and pharmaceuticals such as prozac and ritalin that affect serotonin and dopamine systems and 2) retinoic acid effects upon neural tube development (Dobbs-McAuliffe et al. 2004, Mech. Dev.; Deak et al, 2005, Birth defects research; Zhao et al. 2005, Gene exp patterns) and how the non-liganded retinoic acid receptors play a role in attracting corepressors and histone deacetylase activity to repress genomic regions in the early embryo.
Both of these areas of research have used microarray analysis. Through Duke's NIEHS funded Toxicogenomics Genomics Research Consortium and Duke's NIEHS funded Superfund Center, we have developed a 500+ microarray database of Agilent 22k zebrafish array data. Through the Toxicogenomics Consortium we developed the first 22k array which eventually became a commercial Agilent product. We now use these in examining a variety of events occurring during development in both of our two directions of research. When David Schwartz left Duke to assume the directorship of NIEHS, I became director of an NIEHS funded Toxicogenomics Research Consortium.
Where are we at now?
Through working with the laboratory of Dr. Edward Levin, a psychologist who has development various behavioral and learning tools for zebrafish, we have shown that very early exposure of zebrafish embryo to the pesticide Dursban (chemical name, chlopyrifos) it inhibition of acetylcholine esterase within the first 5 days of development results in later adult learning deficiencies (Levin et al. 2003, 2004, Neurotox. Teratol.). We have preliminary data that strychnine (a glycine receptor antagonist) also produces such results, and Prozac, a serotonin uptake inhibitor, also produce such results. Therefore, we are concerned that the developing nervous system, if exposed to compounds that affect neuronal signaling, may be permanently affected. From a human standpoint, fetal meconium studies from women who live/work in agricultural areas of the Philippines indicate levels of such pesticides and metals. We are concerned that children are being purposely given Prozac and ritalin.
To try to isolate the effects of these compounds, we are using transgenesis in two ways: 1) we have introduced a light activateable chloride pump into the nervous system as a fluorescent fusion protein--this will allow us to turn off neuronal activity with light--to segregate neuronal activity changes from other side effects of exposing animals to chemicals; and 2) we are making transgenic lines of zebrafish with fluorescent reporter genes of neuronal activity in different neurotransmitter pathways to determine whether we might have the sensitivity to use these lines to screen chemicals for their ability to affect neuronal activity in the embryo.
Our retinoic acid work is focusing upon a little noticed function of the retinoic acid receptors, that the receptors, in the absence of the ligand retinoic acid, assemble on retinoic acid response elements in promoters and attract corepressors that attract histone deacetylases to repress the chromatin region around the response elements. We believe this is an important function of the receptors in the early embryo and in stem cells and are exploring, through a number of techniques the possible direction repression of genes through this molecular mechanism and the intersection of this process with other repression systems that are known to maintain the undifferentiated, pluripotential aspect of embryonic stem cells. Microarray work with retinoic acid induced zebrafish embryos induces a series of genes now know to be up-regulated during the differentiation of human embryonic stem cells. We are examining potential mechanisms and pathways for this possible interaction.