R David Heathcote - US grants

The long-term goal of this project is to understand how cell division is regulated to produce the appropriate type and number of neurons within a specific ganglion of the peripheral nervous system. In particular, the project will address the position and timing of neuronal precursors as they divide to form differentiated neurons. These aspects of neurogenesis will be examined in the cardiac ganglion of the frog Xenopus laevis. By concentrating on this extremely simple ganglion, we hope to obtain information that will guide us in understanding neurogenesis in more complex portions of the nervous system. Preliminary experiments have shown that neurogenesis occurred within cardiac ganglia that were isolated in culture. Organ culture, DNA labeling, and autoradiography will be used to determine if neurogenesis within the ganglion is restricted to this short period of time or if it continues throughout the prolonged period of neuron differentiation. Since cardiac neurons are matched to the size of the heart throughout development, the role of heart tissue in stimulating the division of neuronal precursors will be examined in culture. By applying short pulses of labeled analogs of DNA bases to normal developing animals, the length of the S (DNA synthesis) phase of the neuronal cell cycle will be measured. The length of the S phase, together with data on the length of the total cell cycle, will be used to determine the rate of neuronal cell division. This rate of mitotic activity will then be compared with the rate of neuron differentiation at different stages of development.

[unreadable] DESCRIPTION (provided by applicant): The objective of this proposal is to understand how a novel action of estrogen and man-made xenoestrogens affect the initial patterning and development of the nervous system. This will expand our knowledge of the signaling pathways that regulate a specific type of neuron. It also identifies a potential cellular target of endocrine disruptor molecules, which may be related to long-term effects on organismal development. These studies utilize Xenopus laevis, an NIH model organism. The first specific aim tests whether the amount and location of estrogen plays a role in the normal development of dopaminergic neurons and if exogenous estrogen and xenoestrogens are localized near the affected neuron population. The second aim identifies the molecular signaling pathway utilized by estrogen to affect dopaminergic neurons through loss- and gain-of-function experiments with different estrogen receptors. These experiments use antisense morpholinos to knock down specific estrogen receptors and the RNA of individual receptors to achieve their overexpression. The effects will be measured on the dopaminergic neuron population during normal development and in response to additional estrogen. The third aim tests the long- term consequences of brief embryonic exposure to estrogen and xenoestrogens. Prolonged exposure to estrogen during later periods of development affects sex determination and metamorphosis in Xenopus. Given the pronounced cellular effect on dopaminergic neurons after brief embryonic exposure, they could mediate these developmental changes. If estrogenic molecules including Bisphenol A and Octylphenol can alter sex determination and metamorphosis in developing Xenopus, it would indicate there are potential hazards associated with embryonic exposure to estrogenic compounds in a wide range of vertebrates, including humans. Relevance to public health. Endocrine disrupters are man-made molecules present in the environment that interfere with the normal hormonal signaling pathways in humans and animals alike. This proposal examines the molecular mechanisms used by estrogen and endocrine disrupters that mimic it (xenoestrogens) to affect the initial development of a specific population of neurons and its potential long term effects on the organism. [unreadable] [unreadable]