Ronald Booker - US grants

The major objective of this research project is to determine how cell lineage, developmental hormones, cell interactions and developmental genes influence the fate of neurons within the developing nervous system of an insect. Within the nervous system of the moth, Manduca sexta , adult specific neurons are generated during the larval stage by arrays of neuroblasts. Each neuroblast generates a nest of developmentally arrested progeny. With the onset of metamorphosis the development of the adult specific neurons resumes. Electrophysiological techniques will be employed to determine both the physiological and morphological properties of the arrested cells during the larval stage and as they develop into mature neurons. The influence of the developmental hormones on the fate of the new neurons will be examined. Using surgical manipulations, Dr. Booker will also determine if the size of the periphery influences the fate of the adult specific neurons. The role of segmental determination in the development of the insect nervous system will be examined using two approaches. First, a screen for new homeotic mutations in the moth will be conducted. The new mutants will be analyzed to determine if they influence the fate of identifiable pattern elements within the segmental ganglia. Second, DNA in Manduca which shares homology with the homeobox genes of Drosophila melanogaster will be cloned. The cloned moth genes will be characterized as to the temporal and spatial pattern of their expression within the segmented ganglia of both wild.type and mutant animals. These complimentary approaches will further the understanding of the role of the developmental genes in segmental and neuronal determination.

Schneiderman 9422143 During the development of the neuromuscular system, a series of complex interactions are involved in the specification of the pattern of innervation of any specific muscle. This research is designed to elucidate the sequence of events that result in the development of the appropriate connections between neurons and muscles. To address this problem a novel invertebrate system will be employed. The innervation of muscles will be studied during the process of metamorphosis, when degenerating muscles are being replaced with newly formed muscles. In particular, the role of hypothesized "muscle pioneer cells" in neural specification will be examined. The results of this research will provide basic information about the major steps involved in the development of connections between neurons and muscles, as well as insight into the plasticity inherent in the developing and regenerating neuromuscular system.

9729581 BOOKER During development of an organism, more cells are formed than are needed for the final body plan. This excess cell formation is particularly noteworthy in the nervous system, where individual cells have unique roles. Programmed cell death (PCD) plays a central role in the elimination of excess cells and in sculpting developing systems. The goal of this project is to improve our understanding of the cellular mechanisms underlying PCD, using the central nervous system of the moth Manduca sexta as a model system. Programmed cell death is a common feature of Manduca's postembryonic development. During the larval stage, thousands of adult-specific neurons are added to the ganglia of the central nervous system, and throughout this period of cell addition, cell death is also observed. In addition, the transition between the larval and adult stages is characterized by the death of obsolete larval muscles and neurons that are not required by the adult. Previous research has shown that the pattern of cell death observed in Manduca depends on such things as the cell's lineage, segmental identity, and time of birth. This complex pattern of cell death most likely results from differences in the mechanisms underlying PCD in different cells. Dr. Booker will carry out a series of experiments that are designed to investigate these mechanisms. A series of endocrine manipulations will be used to determine whether the steroid hormone ecdysone plays a role in regulating the neuronal PCD observed in the larval stage, and after the onset of metamorphosis. During postembryonic development, the level of cell death is higher in the abdominal compared to the thoracic ganglia. To learn more about the role of segmental identity in the regulation of PCD, a moth homeotic mutant, in which one thoracic segment is duplicated, will be examined to determine if its pattern of cell death is altered. A biochemical characterization of PCD within the nervous system will be done using a series of specific inhibitors of the ubiquitin-dependent proteolytic pathway, because preliminary evidence suggests that this pathway plays a key role in PCD in the moth. Dr. Booker's group has found that application of potent inhibitors of the ubiquitin-dependent pathway can selectively block the PCD of muscles in the developing moth. This finding will allow them to directly test which cell types, at which developmental stages, utilize this mechanism of PCD. The results obtained from this series of experiments will improve our understanding of the biochemical mechanisms involved in PCD, and how PCD can selectively occur in different cell types at different times.

The nervous system of all animals is composed of cells called neurons. These neurons communicate with each other by releasing (secreting) packets of chemicals called neurotransmitters and neuropeptides which signal other neurons. This interneuronal communication is the way the nervous system processes information, and modulation of this process underlies learning and memory. The release of neurotransmitters and neuropeptides is thought to be controlled by a set of proteins called the SNAREs. Just how these SNARE proteins function has remained elusive despite intensive study. The SNARE proteins are highly conserved in all animals and similar proteins have even been identified in yeast. Thus, by studying SNARE function in the fruit fly Drosophila melanogaster, the function of the SNARE proteins can be applied to the secretory process in all animals. With the use of selected SNARE mutants, transgenic expression of SNARE genes in Drosophila, and a novel neuropeptide release assay, the function of these proteins in neurotransmitter and neuropeptide release will be determined.

Elucidating the process of neurotransmitter and neuropeptide release is essential for understanding behavior. More complex functions in the nervous system such as learning and memory involve alterations in synaptic strength. One major way of altering synaptic strength is by modulating the process of neurotransmitter release. Without a clear understanding of the fundamental mechanism of neurotransmitter release (the goal of this proposal), there is little chance of truly understanding the fascinating nature of learning and memory. Neuropeptides have other crucial functions in maintaining organismal homeostasis. Water balance, reproductive behavior, thirst, hunger, metabolic regulation, stress, sleep, and circadian rhythms are all controlled by neuropeptide release.

Neuropeptide secretions from the nervous and endocrine systems are used to precisely coordinate organ systems within the animal so that they function together, leading to a state of balance. Thus, determining how neuropeptide release is governed will greatly improve our understanding of how all animals regulate these complex organ systems. In light of evidence that environmental pollutants are capable of disrupting endocrine function by mimicking hormones, the studies in this proposal are crucial for defining normal endocrine function. These studies will also contribute to the defining the basic process of secretion on which all eukaryotic cells need for survival. The results of this work will be published in widely read journals for scientists and the scientific materials produced will be readily available to the scientific community. In fact, one such strain has already been donated to the Bloomington Drosophila Stock Center, making it readily available to all researchers. Furthermore, at Cornell University, two courses will include material from these studies to extend the educational goals of this research. Cornell's diverse student body ensures that scientific training will extend to underrepresented minorities in the sciences.