Robert J. Denver - US grants

Developing animals respond to variation in their habitat by altering their rate of development and/or their morphology; i.e., they exhibit developmental plasticity. One mechanism by which plasticity is expressed is through activation of the neuroendocrine system, which transduces environmental information into a physiological response. Recent findings of ours with amphibians and others with mammals show that the primary vertebrate stress neuropeptide, corticotropin-releasing hormone (CRH) is essential for adaptive developmental responses to environmental stress. For instance, CRH dependent mechanisms cause accelerated metamorphosis in response to habitat desiccation stress in some amphibian species, and intrauterine fetal stress syndromes in humans precipitate preterm birth. The objectives of the proposed research are to analyze the cellular and molecular mechanisms of CRH signaling during vertebrate development. Tadpoles of the South African clawed frog Xenopus laevis will be used as the model organism. The results of these studies will provide basic information on the molecular and functional organization of this essential developmental regulatory system. Furthermore, these studies should lay a foundation for understanding the mechanistic basis of developmental responses to changes in the physical and chemical environment. Basic knowledge gained from these studies should allow a more informed and detailed understanding of normal pathways of endocrine control, knowledge that will be essential to elucidating the mechanisms by which environmental endocrine disrupters act to alter developmental processes. Such a goal is particularly cogent given recent reports of worldwide declines in amphibian populations and concerns over the effects of hormone mimicking industrial compounds on the development of humans and wild animals.

PI: Denver, R. IBN-9724080 Normal development of the vertebrate brain requires thyroid hormone (TH). Insufficient amounts of hormone during fetal life results in severe defects in neural development (e.g., cretinism in humans). On a global scale, congenital hypothyroidism is one of the most pervasive and detrimental human health problems, resulting in entire populations of mentally impaired ad physically stunted individuals in certain areas of the world. In addition to hypothyroidism caused by iodine deficiency, recent reports of disruptive effects on the thyroid axis of various industrially-produced compounds suggest the possibility for profound negative effects of environmental pollutants on developmental processes of humans and wildlife. The proposed research aims to understand the molecular basis of TH action on the development of the vertebrate central nervous system (CNS). Tadpoles of the African clawed frog, Xenopus laevis will be used to study TH-induced neural development and to clone TH-target genes in the CNS. This model in invaluable for understanding post-embryonic brain development since it affords easy access to all stages of development, and the basic neurodevelopmental processes are similar in frogs and other vertebrate animals including humans. The primary objectives are to: 1) understand how TH receptors transduce the hormonal signal to the cell nucleus resulting in changes in gene expression, and , 2) understand the structure and function of TH target genes in the CNS.

Normal brain development is critically dependent on thyroid hormone (TH) which influences neuronal maturation, neurite outgrowth, synapse formation and myelination. While we know much about the gross morphological defects of the central nervous system (CNS) and the clinical manifestations that result from fetal and neonatal hypothyroidism (i.e., severe mental retardation--cretinism), very little is known about the molecular mechanisms of TH action in neural development. This lack of information hinders our ability to fully understand normal development of the CNS, the etiology of developmental disorders of the CNS and to develop effective treatments for such disorders. The primary objective of this project is to provide a molecular basis for understanding TH action in brain development by studying the transcriptional regulatory networks induced by the hormone. The effects of TH are mediated by the action of specific nuclear receptors (TRs) that function as ligand-activated transcription factors. In the proposed work, two models for vertebrate neural development, the rat and the frog (Xenopus laevis) will be used to identify and analyze the function of novel TH target genes in the developing brain. We previously isolated cDNAs for 34 TH-response genes from tadpole brain and conducted cross-species hybridizations to identify three similarly- regulated genes in embryonic rat brain. In the proposed work, the regulation of gene expression and the function in neural development of two genes identified by this approach will be studied. The first aim focuses on the basic transcription element binding protein (BTEB), a GC- box transcription factor whose mRNA levels are strongly regulated by TH in developing rat brain. The regulation of expression of this gene in neural cells will be analyzed at both the transcriptional and posttranscriptional levels. The second aim will analyze the expression and function in neural development of the HMG-box transcription factor HBP1 which has been shown to interact with members of the retinoblastoma family of growth suppressors and likely plays an important role in cell differentiation. The third and final aim will utilize three other cDNA clones for Xenopus TH-response genes (chosen for their hormonal responsiveness and developmental pattern of expression) to identify other novel hormone-regulated genes in rat brain. These two models provide a powerful, complementary system for identifying and characterizing important neurodevelopmental genetic pathways and thus place us in a unique position to provide a molecular basis for understanding both normal and abnormal neurological development.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Exposure to stress early in life can have profound effects on physiology and behavior later in life, which may alter the ability of individuals to grow, compete for resources and reproduce, or may predispose them to disease. The effects of stress during early development are likely to be mediated by elevated levels of stress hormones (glucocorticoids) that serve to 'program' gene expression in the brain and other organs, leading to stable, long term changes in physiological function. This research is investigating the roles that glucocorticoids play in shaping the development of the neuroendocrine stress axis, which plays a key role in controlling behavior and physiology. For this work the investigators are using tadpoles and juveniles of the frog Xenopus tropicalis. The research is multidisciplinary, relying on immunohistochemical, endocrine, and behavioral approaches, and state-of-the-art molecular genetic and genome-wide analyses. This research will provide basic knowledge of the physiological and molecular mechanisms by which early life stress alters later life physiology and behavior in vertebrates. This work is also important for understanding the impacts of environmental degradation on the health of amphibian populations. Environmental insults during the tadpole stage can lead to lasting effects that could impact adult fecundity and survival, and thus contribute to population declines and extinctions. Understanding this potential, and the mechanisms involved is essential to devising effective conservation strategies. This grant supports the training of a postdoctoral scientist, a woman Ph.D. graduate student, and provides opportunities for undergraduate honors students to participate in research that will encourage the pursuit of careers in science.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of the proposed studies is to understand basic mechanisms of thyroid hormone (T3) action in brain development. Brain development is critically dependent on T3, which promotes axonal maturation, dendritic arborization, synapse formation, myelination, cell proliferation and apoptosis. Thyroid hormone deficiency during neonatal/fetal life results in severe mental retardation (i.e., cretinism). Despite the profound effects of thyroid deficiency, relatively little is known about the molecular mechanisms of T3 action in CMS development. Electrical activity in the CNS promotes the elaboration of axons and dendrites, and is required for the establishment and strengthening of synaptic connections. Thus, both hormones and electrical activity are necessary for normal development of the vertebrate brain. Analysis of the immediate early genes (lEGs) induced by electrical activity and by T3 in the developing CNS suggests overlapping signaling pathways. Earlier we discovered that the transcription factor basic transcription element binding protein (BTEB1) is strongly upregulated during postnatal development in rodent brain. We showed that BTEB1 is directly regulated by T3 and that BTEB1 plays a role in neuronal morphogenesis, stimulating neurite extension and branching. Our findings support the view that BTEB1 is a critical player in T3-dependent neuronal morphogenesis. In addition to hormonal regulation, we recently found that BTEB1 is an activity-regulated gene in the CNS. To elucidate the role that BTEB1 plays in mammalian brain development, we propose to: 1) analyze developmental and hormone-dependent BTEB1 expression in mouse CNS, 2) analyze thyroid regulation of the mouse BTEB1 promoter, and 3) analyze activity-dependent regulation of the mouse BTEB1 gene. This research will provide an important foundation for understanding the molecular basis of T3 action on brain development, and will begin to integrate knowledge of hormone-dependent signaling pathways with electrical activity-dependent pathways. A detailed knowledge of these pathways is necessary for understanding basic neurodevelopmental processes, and may suggest strategies for prevention and/or treatment of neurological disorders caused by disruption of hormone signaling pathways. [unreadable] [unreadable]

Leptin, the protein product of the obese gene, is a hormone secreted by fat cells that is integral to food intake regulation in mammals. Leptin signals to the brain information about long term energy balance, and thus influences critical aspects of the life cycle such as growth and reproduction in many species. Additionally, the prevalence of obesity in developed countries in recent years has focused intense interest on leptin and other factors that influence appetite and energy metabolism.
Dr. Denver's lab reported the first definitive identification of the obese gene and the functional characterization of leptin in a nonmammalian species, the South African clawed frog, Xenopus laevis. Until now, virtually nothing was known about the biology of leptin outside of mammals. The overall goal of this research is to understand the functions of leptin in a cold blooded species, Xenopus laevis, whose lineage diverged from that of modern mammals over 200 million years ago. The frog has been, and continues to be an important model organism for the study of animal development. Using molecular, physiological and developmental approaches, the major questions to be addressed in this research are: 1) Does leptin play an evolutionarily conserved role in long term energy balance in frogs, and thereby influence critical aspects of the amphibian life history such as metamorphosis, growth and reproduction? 2) Where and when in the frog's body is leptin produced, and where are leptin's major sites of action? 3) Does leptin influence tadpole brain development, alter tadpole growth and the timing of metamorphosis?
This project, which will offer unique training opportunities for undergraduate and graduate students, will provide a foundation for understanding the functional evolution of this important vertebrate hormone, and establish the frog as a model system for the study of leptin actions in early development. Basic research on the hormonal control of appetite and feeding is particularly timely given the global concern over the rising incidence of obesity and related disorders in humans.

Two international scientific meetings will be supported at the University of Michigan in July, 2011: the inaugural meeting of the North American Society for Comparative Endocrinology (NASCE 2011), and the 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology (ISAREN 2011). The NASCE is a new scientific society formed in 2010 with the purpose of promoting the comparative study of hormones and hormone action. This includes topics in evolutionary, environmental (including endocrine disrupters), general and biomedical endocrinology/neuroendocrinology. The ISAREN was formed in 1992 to promote the study of the endocrinology and neurobiology of amphibians and reptiles.

The NASCE 2011 and ISAREN 2011 meetings will revitalize and strengthen the field of comparative endocrinology in North America by bringing together students, young investigators, and trainees from diverse areas and backgrounds to exchange ideas and to establish and strengthen collaborations. We promote diversity in our field of science as well as diversity of scientific topics in the meeting program, and we provide significant opportunities for groups traditionally underrepresented in science to attend the meeting. Meeting abstracts will be published in the open access journal Frontiers in Endocrinology, and the plenary lectures and selected symposium presentations will be published in the journal General and Comparative Endocrinology. This will allow for the presentations to be widely disseminated to the scientific community.

The goal of this project is to advance understanding of brain development by studying a novel role of thyroid hormone (TH) in the regulation of gene expression in early development. It is known that the expression of genes is controlled in part by chemical modifications to deoxyribonucleic acid (DNA) and the proteins that surround the DNA. These so-called 'epigenetic modifications' result in heritable changes in gene function that do not involve changes to the DNA sequence itself. Recent findings show that epigenetic modifications play pivotal roles in organismal development, physiology, and behavior, and are implicated in human diseases from cancer to complex behavioral disorders, such as autism and schizophrenia. One such epigenetic modification is the addition of methyl groups to cytosine residues in DNA (DNA methylation). Variable DNA methylation at gene regulatory regions can influence gene expression. DNA methylation typically promotes gene repression, whereas DNA demethylation promotes gene activation. However, the patterns and functions of DNA methylation in the brain during development, and the factors that control DNA methylation are poorly understood. The Principal Investigator (PI) recently discovered that TH, which is known to be critical for normal development of the central nervous system, controls DNA methylation in the developing brain. Here, he will examine the mechanism of DNA methylation by TH and its consequences for neurological development using the frog as model system. The project also will involve training of a diverse scientific workforce, establishment of an undergraduate course in experimental design for life scientists, and science outreach activities with middle school students and with adults at retirement communities.

The specific aims of this project are to: 1) map DNA methylation patterns in the developing brain using genome-wide analyses including RNA sequencing, chromatin immunoprecipitation sequencing, and methyl capture sequencing, 2) determine the mechanisms by which TH regulates DNA methylation using targeted analysis of TH-dependent recruitment of enzymes to chromatin, and 3) understand the consequences of TH regulation of DNA methylation for neurological development through analysis of cell cycle control. The PI will focus on metamorphosis of the frog Xenopus (Silurana) tropicalis, a well studied TH-dependent process. They will conduct genome-wide methylation and transcription factor association analyses to investigate the patterns and roles of DNA methylation during postembryonic brain development. The results of the proposed research will provide a deeper understanding of factors that modulate the epigenetic landscape in neural cells, and will open new avenues for research on the regulation and roles of DNA methylation in neurological development and function.

? DESCRIPTION (provided by applicant): Epigenetic modifications, which result in heritable changes in gene function that do not involve changes to the DNA sequence, play central roles in human brain development. Dynamic regulation of DNA methylation by de novo DNA methyltransferases (Dnmts) is implicated in the control of neuronal and glial cell differentiation. We recently discovered that the Dnmt3a gene is a direct thyroid hormone (TH) receptor (TR) target in mouse brain. Postembryonic brain development is critically dependent on thyroid hormone (TH); TH deficiency during fetal and neonatal periods of human development leads to a condition of severe mental and growth retardation known as cretinism. We hypothesize that TH regulation of Dnmt3a plays a pivotal role in normal neurological development, since recent evidence supports that dynamic postnatal regulation of Dnmt3a may be essential for establishing DNA methylation patterns in the developing brain. In the proposed research we will: 1) Investigate the regulation of the Dnmt3a gene in mouse brain in vivo by TH throughout early postnatal development. We will investigate TR recruitment to the Dnmt3a locus, and histone modifications induced by TH. We will directly test whether TH response elements that we identified are required for TH regulation of the Dnmt3a gene. 2) Investigate a role for Dnmt3a in TH-dependent neural cell cycle arrest and differentiation. We will investigate whether cell cycle arrest is mediated by Dnmt3a methylation of the promoter of the cell cycle control gene Cyclin D1, and possibly other E2F target genes. For these experiments we will use mouse neuronal cell models that expresses the ß1 isoform of TR. Proper exit from the cell cycle, cell differentiation and the maintenance of cell cycle arrest is essential for normal development. Disruption of these processes in the embryo and fetus can lead to abnormal development; disruption in the adult can lead to cancer and other disease. Thyroid hormone plays a central role in cell cycle exit and cell differentiation in the central nervous system. Several lines of evidence support that Cyclin D1 is directly regulated by Dnmt3a-mediated DNA methylation. We hypothesize that TH regulation of DNA methylation of regulatory regions of cell cycle control genes plays a key role in this process. Successful completion of the proposed research will lead to advances in our understanding of the mechanisms by which DNA methylation is regulated during development, and the roles of TH-dependent DNA methylation in cell proliferation and differentiation.

Predator-prey interactions are a common feature of ecological systems and have been shown to drive their structure, function, and dynamics. Even so, critical knowledge gaps exist with respect to the mechanisms that underlie the prey's behavioral, physiological, and morphological response to predation risk, including how those responses influence the performance of predator. This study will use a model system (predatory larval dragonflies and tadpole prey) to understand how predation risk and food availability modulate the anti-predator response. This study will build upon a large body of experimental research that shows that the fear response of the tadpoles governs both their behavioral (e.g., activity) and morphological (e.g., tail growth) responses to predators. The approach taken will be integrative and interdisciplinary, with endocrinology measurements used in conjunction with laboratory experiments and computational models of optimal prey responses. This study will include K-12 classroom visits, the development of new college courses, and the provision of student research opportunities all aimed at improving the preparedness of students, especially under-represented groups, for entering and succeeding in STEM fields.

The specific aims of this research are to determine: 1) how the prey neuroendocrine stress response operates over time and is shaped by complex predation environments and trade-offs; 2) how stress hormones govern the expression and integration of the prey phenotypic response (i.e., behavior, morphology) in an ecological context; and 3) how the prey phenotype influences predator-prey interactions. The intellectual merit of this proposal centers on our use of a model ecological system, novel experiments, and novel optimality modeling to shed insight into how physiology and development regulate plasticity in adaptive traits and what the ecological impacts of this regulation are. Identifying the proximate physiological mechanisms that govern phenotypic expression will enhance the general understanding of the phenotypic range that prey are capable of achieving in response to predators, the tradeoffs that influence costs and benefits of particular phenotypes, and how predators induce prey phenotypes. Identifying the mechanisms underlying prey phenoptypic plasticity also will help clarify the role of nonconsumptive effects in ecological communities. Ultimately, the findings generated herein will provide important insight into the linkages that exist among the environment, an organism's physiological response and its resulting phenotypic plasticity, and the fitness consequences to both prey and predator.