MICRORNAS PLAY CRITICAL ROLES IN DEVELOPMENT RESPONSES TO DIETARY CONSTITUENTS
Robert Tanguay, Ph.D.
Summary: MicroRNAs—non-coding RNAs that affect gene regulation—have emerged in recent years as important regulators of many cellular and physiological processes. In zebrafish, microRNAs prevent excess vitamin A from causing spine defects during development. Exposure to alcohol during early development in zebrafish interferes with microRNA activity, resulting in abnormalities in the development of the nervous system.
It is well established that dietary constituents can either positively or adversely affect fetal development, but we donít have a good understanding of the relevant mechanisms. As you may remember from biology class, there are several forms of RNA. During a process called transcription, enzymes produce complementary RNA from DNA. Messenger RNA transfers information from DNA to the cell's ribosome, where proteins are made. Over the past ten years, another kind of RNA—non-coding RNA (ncRNA)—has emerged as a pivotal player in fundamental physiological and cellular processes and has been increasingly implicated in cancer, immune disorders, and cardiovascular, neurodegenerative, and metabolic diseases. MicroRNAs (miRNAs) are a class of ncRNA molecules that are predicted to post-transcriptionally regulate the expression of 30-60% of all human protein-coding genes, and as such, microRNAs play key roles in cellular and developmental processes. MicroRNAs have emerged as targets of developmental, hepatic, neurological, and carcinogenic toxicological agents and have increasingly been identified as regulators of xenobiotic-metabolizing enzymes. The goal of our pilot study funded by LPI was to determine whether misregulation of miRNA expression during development constitutes a specific mechanism by which developmental toxicants exert their effects.
Vitamin A is necessary for normal vertebrate growth and development. Jill Franzosa, a graduate student in my laboratory, investigated the developmental role of miRNAs using an active form of vitamin A, all-trans retinoic acid (RA). We know that excesses or deficiencies of RA adversely affect development. In this study, we demonstrated that treating zebrafish in early developmental stages with RA results in a distinct curved body axis similar to the axis and spine defects observed in other vertebrates, including humans. To determine whether specific miRNAs are misexpressed after RA exposure, gene analyses were conducted at several critical developmental stages. Strikingly, the expression of three miRNA family members MicroRNAs Play Critical Roles in Development Responses to Dietary Constituents was significantly repressed by RA treatment during the early stages of somitogenesis (development of skin and skeletal muscle and bones). Based on a prediction that miRNAs target the key RA-inactivating enzyme, cyp26a1, we then confirmed, for the first time, that cyp26a1 is a bona fide target of the specific miRNA family. We also showed that repression of the implicated miRNA by genetic alterations resulted in similar embryonic defects. When specific miRNA mimics were injected into embryos, axis defects induced by developmental exposure to RA were prevented. Together, these results indicate that the axis defects elicited by exposure to RA during development result partly from repression of specific miRNAs and subsequent misregulation of cyp26a1. This study sheds light on the role of miRNAs in mediating the teratogenic effects of retinoic acid and provides a more in-depth view of the genetic regulatory mechanisms that control the action of vitamin A during normal development.
In other studies, postdoctoral fellow Tamara Tal used alcohol as a neurotoxicant in zebrafish to examine the role of miRNAs in the development of a functional nervous system. While miRNAs are critical to nervous system development, the neurobehavioral function of miRNAs is unknown. Prenatal alcohol exposure produces a range of conditions in offspring, collectively referred to as fetal alcohol syndrome disorder (FASD). Epidemiological studies suggest that the majority of children with FASD have deficits in neurobehavioral function, even in the absence of clinically discernable physical abnormalities. We assessed larval zebrafish neurobehavior by measuring the distance moved during alternating periods of light and dark. Transient exposure to alcohol during early neural development resulted in increased physical activity. These alterations in behavior persisted in juveniles that had been developmentally exposed to alcohol.
At developmental stages in zebrafish coincident with alcohol-induced neurobehavioral abnormalities, expressions of multiple miRNAs in the central nervous system were significantly altered. Subsequent computational analyses revealed that alcohol disrupts expression of miRNAs that direct neurogenesis.
We then used genetically altered zebrafish to examine the functional role of alcohol-sensitive miRNAs. Decreasing the activity of miRNAs in the central nervous system produced behavioral hyperactivity in larval and juvenile fish similar to that observed with alcohol. These data indicate that ethanol exposure causes errors in the expression of miRNAs that may collectively choreograph nervous system development and function and support the concept that miRNA signaling pathways are targets of developmental neurotoxicants like alcohol.
Our research resulted in numerous awards and recognitions. Jill Franzosa's research was honored with the Elsevier best platform presentation award in toxicogenomics at the 2010 Society of Environmental Toxicology and Chemistry annual meeting, first place poster award at the 2010 Pacific Northwest Association of Toxicologists Conference, poster award winner at the 2010 Aquatic Animal Model for Human Disease Annual Meeting, first place in the Molecular Biology graduate student award at the 2010 Society of Toxicology annual meeting, and a first place platform presentation award at the 2009 Pacific Northwest Association of Toxicologist Conference.
Dr. Tamara Tal's research in alcohol-sensitive microRNAs was recognized at the 2010 Pacific Northwest Association of Toxicologists Regional Conference, won a first-place poster award at the 2010 International Neurotoxicology Conference, won a first-place postdoctoral research award from the Molecular Biology specialty session, and won a second-place poster award from the Neurotoxicology specialty section at the 2010 Society of Toxicology annual meeting. Both Dr. Tal and Jill Franzosa recently presented their research in the symposium session titled "Uncovering the role of microRNAs in toxicology" at the 2011 Society of Toxicology annual meeting.
Based upon the success of this project, Jill was also awarded a prestigious predoctoral National Institutes of Health Ruth L. Kirschstein NRSA Fellowship to investigate the role of microRNAs in aging. Together, these findings will generate new fundamental knowledge about the role of microRNAs in mediating basic biological responses to chemical or dietary toxicological insult.
Last updated July 2011