Patrick Larkin

Differential gene expression analysis in fish exposed to endocrine disrupting compounds

This review discusses various methodologies that can be used to understand, at the gene level, the consequences to fish upon exposure to endocrine disrupting compounds (EDCs). Several approaches for measuring expression of gene transcripts are discussed, including directed approaches, such as Northern blotting and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) as well as open-ended approaches, such as differential display RT-PCR, subtractive hybridizations, and gene arrays. Each of these systems has advantages and disadvantages, strengths and weaknesses. Conducting experiments with each of these methods provides important information about the molecular mechanisms that result from exposure to EDCs, information which can be used in risk assessment of polluted sites found in the environment.

Circadian regulation of iodopsin and clock is altered in the retinal degeneration chicken retina

We are interested in determining if the visual phototransduction cascade plays a role in light entrainment of photoreceptor circadian oscillators. In this study, we compared mRNA levels of iodopsin and the chicken homolog of Clock (cClock) in the retinas of normal and rd (retinal degeneration) chickens that lack functional rod and cone phototransduction cascades. Iodopsin is a circadian-regulated, photoreceptor-specific gene expressed in chicken retina, and Clock is a transcription factor that has been shown to play a role in the circadian clock mechanism in mouse and Drosophila. The results of our analyses show that cClock and iodopsin transcript levels undergo daily oscillations in retinas of normal animals housed under 12 h light:12 h dark (12L:12D) conditions, and that these oscillations are maintained in the absence of light. Levels of these transcripts in the retinas of rd/rd chickens housed under cyclic light conditions did not change significantly over the course of a 12L:12D cycle; however, there was evidence that the photoreceptor oscillators were entrained in these animals. Comparisons of our normal and rd/rd data suggest that there are at least two light entrainment pathways that impinge on the oscillators found in photoreceptor cells, one of which is effectively disabled by the GC1 null mutation carried by the rd chicken.

DEVELOPMENT AND VALIDATION OF A 2,000-GENE MICROARRAY FOR THE FATHEAD MINNOW (PIMEPHALES PROMELAS)

Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17beta-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17beta-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiols expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.

Array technology as a tool to monitor exposure of fish to xenoestrogens.

A variety of anthropogenic chemicals are capable of binding to the estrogen receptor of vertebrate species. Binding of these compounds can interfere with homeostasis by disrupting normal gene expression patterns. The purpose of this study was to investigate the feasibility of applying array technology as a monitoring tool for detecting the presence and distribution of estrogenic compounds in coastal habitats using sheepshead minnows as our model. cDNA clones that were isolated from differential display, including vitellogenin alpha and beta, vitelline envelope protein (ZP2), and transferrin, among others, were spotted on the macroarray. The results of these experiments demonstrate a characteristic expression pattern of estrogen responsive genes in sheepshead minnows exposed to 17 beta-estradiol (E2).

Chapter 3 Approaches in proteomics and genomics for eco-toxicology

Publisher Summary This chapter reviews current methods to study the transcriptome and the proteome (the set of proteins encoded by a genome) and indicates approaches that can be taken for non-model species. The chapter briefly explains genomics and proteomics. The ultimate goal of genomic experiments is to link-induced gene expression patterns to detrimental effects, harmless effects, or even protective effects. The term “proteomics” refers to the large-scale study of proteins in cells or tissues and also understanding the complex interactions among proteins that occur within cells. These interactions include formation of functional complexes as well as interactions with other cellular components such as nucleic acids, lipids, and carbohydrates. Proteome research has been subdivided into two categories: (1) expression proteomics and (2) interaction proteomics.

A null mutation in guanylate cyclase-1 alters the temporal dynamics and light entrainment properties of the iodopsin rhythm in cone photoreceptor cells

Guanylate cyclase-1 (GC1) plays a critical role in visual phototransduction and its absence severely compromises the ability of the photoreceptor cells to transduce light for vision. In this study we sought to determine if the absence of GC1 has any effect on light entrainment of the circadian oscillators located in these cells. We compared the rhythmic changes in transcript levels of iodopsin, a photoreceptor-specific gene whose expression is regulated by circadian oscillators, in retinas of normal chickens and GUCY1*B (*B) chickens that carry a null mutation in GC1. Our results show that iodopsin rhythms are present in *B retinas and that they can be entrained to light; however, the rise and fall of iodopsin transcript levels in *B retina under cyclic light conditions is significantly more rapid than that observed in normal retina, and under constant dark conditions, the phase of the iodopsin rhythm in *B retina is advanced by 6 h relative to that observed in normal retina. In addition, the rate of entrainment of the iodopsin rhythm in *B retina to a reversal of the light cycle is significantly slower than normal. The results of our study show that a functioning visual phototransduction cascade is not essential for light entrainment of the oscillators that drive the iodopsin rhythm in photoreceptor cells. We propose that the abnormal synthesis of cGMP in *B photoreceptors underlies the irregular iodopsin rhythms observed in post-hatch *B retina.