The proposed studies are designed to test the hypothesis that anterior pituitary hormones act as developmental neurotrophic signals for hypothalamic pituitary-regulating (hypophysiotropic) neurons. The broad, long-term objective of the research is to elucidate the mechanisms by which these endocrine signals affect hypophysiotropic neuron survival, differentiation, and axon terminal guidance. The studies will be conducted using two types of dwarf mouse with spontaneous pituitary transcription factor mutations that result in failure to produce growth hormone (GH) and prolactin (PRL), and which show concomitant abnormalities in neurons that produce GH-regulating somatostatin and GH-releasing hormone, and PRL-inhibiting DA. Thus, the effect of absent signal during development may be assessed without experimentation, and hormone treatments may be selective and specific. The general experimental design is evaluation of developmental events in the absence of target feedback, and of effects of hormone replacement on these events.
The specific aims are to determine, in naive and hormone-treated dwarf mice, 1) the extent to which hypophysiotropic axons terminate aberrantly outside of or within the hypothalamic median eminence (ME) and whether this pattern is regressive, using anterograde and retrograde tract tracing, immunocytochemistry (ICC) and electron microscopy (EM), including assessment of axonal guidance molecules and structural elements in ME, 2) whether programmed cell death occurs postnatally among hypophysiotropic DA neurons, by ICC of apoptotic gene products, nucleosome end-labeling in situ, and EM, and 3) whether IGF-I and GDNF are respective mediators of GH and PRL effects, by assessing expression of these factors and their receptors using in situ hybridization and testing whether either factor can substitute for hormone replacement. Related to the assessment of mediators is Specific Aim 4, further examination of pathways and mechanisms of GH and PRL effect, by localizing GH and PRL receptors, identifying the JAK/STAT proteins that these receptors activate, measuring the expression of immediate-early gene products after GH or PRL treatment, and identifying the neuronal phenotypes showing receptor or activation, because hypophysiotropic neuron stimulation may be indirect.
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