John B. Thomas - US grants

The mechanisms involved in generating the large numbers of precise connections between neurons are unknown. In order to find and eventually synapse with their appropriate target cells, developing neurons choose stereotyped routes, often travelling long distances and bypassing many cells. During insect embryogenesis, the growth cones of individual neurons actively recognize and grow along particular axon surfaces, suggesting the expression of a number of cell-surface molecules mediating these recognition events. The long-term goal of this proposal is to understand the molecular bases of these recognition events. To this end, a most promising approach is to isolate mutations which alter the recognition events and in this way identify the genes and gene products involved. Recently, we have characterized in detail the interaction and recognition between single identified neurons in the Drosophila embryo, with the goal to capitalize on its advanced genetics and molecular biology to extend our analysis to the molecular level. The specific aims of this proposal are 1) to isolate single-gene point mutations in Drosophila which alter the normal stereotyped patterns of recognition between embryonic neurons, 2) to isolate point-mutations, and thus identify the relevant genes, within two chromosomal regions which have been shown to disrupt the patterns of recognition when deleted, and 3) to initiate the molecular cloning of the genes of interest. Mutations will be identified and analyzed by using specific antibodies to the Drosophila nervous system and by Lucifer Yellow dye injections of individual neurons. The molecular cloning of genes will be initiated by obtaining P-element insertional mutations.

The questions addressed in this proposal is a fundamental one in the field of neurobiology, namely how complex neural circuits are assembled during development. Developing neurons of both vertebrates and invertebrats possess the ability to recognize and follow specific pathways, often over long distances, which lead them to their appropriate synaptic targets. While the cell surface guidance molecules involved in these pathway selection events are largely unknown, a combinatorial code of LIM-homodomains (LIM-HD) proteins has been shown to control the axon pathway selection of neurons, likely by regulating the expression of specific guidance receptors. The goal of this project is to identify the guidance molecules regulated by Apterous (Ap), a Drosophila member of the LIM-HD family, as well as to characterize a set of putative axon guidance molecules isolated in a screen for genes that switch the pathway selection of the Ap neurons. Using Drosophila genome arrays, the differences in gene expression between FACS-sorted Ap neurons from wild-type and from ap mutants will be determined at the level of the entire genome. A parallel genetic screen will also be carried out. Misexpression of Ap in a subset of neurons that do not normally express it switches their axon projections to the pathway normally taken by the Ap neurons, supporting the hypothesis that Ap is ectopically regulating genes encoding guidance molecules normally expressed by the Ap neurons. To test this and to identify the genes regulated by Ap, EMS-induced mutations that dominantly suppress the gain-of-function axon-switching phenotype will be isolated and analyzed. From a screen for genes causing pathway switching of the AP neurons, several known and putative axon guidance molecules have been isolated, including the Drosophila ortholog of the Unc5 receptor for the Netrins. These molecules will be tested for their roles in axon guidance by generating mutations in their genes. Given the functional conservation throughout the animal kingdom of many protein families, including the ILM-HD family, these studies will provide us with a better understanding of axon guidance mechanisms not only in Drosophila, but in higher vertebrates as well.

A normal functioning nervous system depends upon the ability of neurons to recognize and synapse with their appropriate target cells during development. Although the cellular events involved in this type of cell interaction have been well described in both vertebrates and invertebrates, the underlying molecular mechanisms are poorly understood. Our long-term goal in this project is to understand the molecular basis of these neuronal recognition events. The approach we have taken is to use the genetics of Drosophila to identify genes and gene products that are involved in neuronal recognition. To this end, we have isolated mutations that alter the synaptic connections between identified neurons within a simple neuronal circuit. One of these mutations, called bendless (ben), appears to alter the recognition event leading to proper connectivity between two well characterized neurons of the circuit, the giant fiber and one of its post-synaptic targets, the TTM motoneuron. We have mapped the genomic location of the ben gene on a molecular walk through the region, and propose both to identify the ben transcription unit and to use molecular genetic and transformation techniques to study the role of the ben gene product in the target recognition process. We will determine which cells require ben function, and also misexpress the gene during development. We expect that these experiments will provide us with a better understanding of the target recognition process and its molecular basis, not only in Drosophila, but in higher vertebrates as well.

[unreadable] DESCRIPTION (provided by applicant): To find and synapse with their appropriate target cells, the growth cones of developing neurons recognize guidance cues in their environment and transduce them into changes in direction of growth. In Drosophila, the binary choice of anterior vs. posterior commissure (AC vs. PC, respectively) made by the growth cones of all neurons that project across the midline, is controlled by Derailed (Drl), an atypical receptor tyrosine kinase, and its ligand Wnt5, a member of the Wnt family of secreted signaling molecules. Wnt5 is secreted by PC neurons and acts as a chemorepellent to keep the Drl-expressing AC growth cones out of the PC. Our goal in this project is to understand how the Wnt5/Drl guidance mechanism functions. We will develop a novel in vitro growth cone turning assay for Drosophila neurons to test whether Wnt5 acts directly on the growth cones of Drl-expressing neurons. Wnt5 is proteolytically cleaved in vivo, but the role this event plays in Wnt5 function is unknown. Using epitope tags, we will determine where and when Wnt5 is cleaved and test the role of cleavage in Wnt5 function. The signaling pathway downstream of Drl that transduces the repulsive signal within the growth cone is unknown. Since Drl is a novel Wnt receptor, we will genetically test whether known components of Wnt signaling, such as Frizzled receptors and Disheveled, function with Drl in axon guidance. To further identify signaling components downstream of Drl, we will determine the identity of two genes that we have shown to strongly suppress Drl function when deleted. In addition, we will express a functional Flag-tagged version of Drl to immunoprecipitate proteins that specifically bind to the Drl cytoplasmic domain and test whether these proteins bind to regions of the Drl cytoplasmic domain required for signaling in vivo. Finally, our work in Drosophila raises the question of how universal the Wnt/Drl axon guidance mechanism might be. We will test the guidance role of Ryk, the mammalian homologue of Drl, by examining motor axon projections in Ryk knockout mice. Relevance: A normal functioning brain relies on the generation of specific connections between nerve cells, as does functional recovery after spinal cord injury. The studies in this project will provide insight into how nerve cells become properly wired. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Gliomas are the most common and deadly malignant tumors of the central nervous system. Glioma cells diffusely infiltrate adjacent and often distant brain structures, a property which renders them largely incurable. To effectively control gliomas, the mechanisms underlying tumor cell invasion and progression must be determined and targeted with novel therapies. However, the signals that govern infiltration are largely unidentified. Mouse models and human tissue culture models have provided valuable insights into the roles of known genetic alterations in glioma, but because of the difficulty in carrying out forward genetic screens, they have not been widely used to discover new genes involved in glioma pathogenesis. The goal of this project is to create a model of glioma in the fruit fly Drosophila for the purpose of carrying out large- scale genetic screens to identify genes involved in glioma invasion and progression. Drosophila glia share many properties with mammalian glia, including the display of infiltrative, glioma-like tumor phenotypes upon co-activation of the EGFR-Ras and PIS kinase signaling pathways. In this project, candidate loci implicated in human gliogenesis will be evaluated and novel regulators of EGFR-Ras/PI3 kinase-induced invasiveness will be searched for. Drosophila orthologs of genes known to be upregulated in glioma, but whose roles in pathogenesis are unknown, will be misexpressed specifically in glial cells and assayed for glioma-like phenotypes. A screen for mutations that enhance or suppress the glioma-like invasive phenotype upon EGFR-Ras and PIS kinase pathway activation will be carried out and the identity of the corresponding genes will be established. The genes identified in these screens will represent excellent candidates for genes directly involved in pathogenesis. Relevance: These studies are expected to provide key insights into glioma pathogenesis, including the identity of genes involved in tumor cell migration and invasiveness. The products of these genes may represent excellent targets for therapeutics. [unreadable] [unreadable] [unreadable] [unreadable]

The CREB family of transcription factors participates in a variety of cellular functions including energy homeostasis. Recently, a new family of CREB coactivators, called TORCs, has been identified. TORCs translocate to the nucleus in response to cAMP and calcium signals, where they potentiate cellular gene expression via a direct interaction with CREB. Drosophila has a single TORC family member, dTORC, which can act as a bone fide member of the TORC family in cell culture coactivation assays. TORC1 +/- mice are hyperphagic, gain more weight than wild type littermates and show hyperinsulinemia. Similarly, dTORC mutant flies show increased food intake, altered glycogen and triglyceride stores, and also show sensitivity to starvation. These phenotypes can be rescued by supplying dTORC in the nervous system, indicating that neuronal dTORC activity is critical for regulation of appetite and energy stores. This proposal aims to use Drosophila to understand how TORC proteins function in these processes. The domains of dTORC that are conserved between mammals and Drosophila will be tested for function. Since dTORC is active in the insulin-producing cells of the fly brain and mutant dTORC flies have phenotypes expected from alteration of the insulin pathway, the hypothesis that dTORC regulates insulin expression and signaling will be tested. Tests for genetic interactions with insulin signaling pathway components will be carried out. The time course of changes in dTORC phosphorylation state and nuclear shuttling in response to feeding and fasting will be examined. In addition to insulin signaling, other components involved in satiety and energy stores are likely to be controlled by dTORC and mammalian TORCs. By capitalizing on a dTORC overexpression phenotype in the eye, a screen for genes that enhance or suppress the phenotype will be carried out. These genes may encode components of the dTORC signaling pathway, and by extension the pathway for mammalian TORCs as well.

ABSTRACT Fasting triggers concerted changes in behavior, physical activity and metabolism that are remarkably well conserved through evolution. In mammals, such responses are often coordinated by transcriptional coactivators that are targets for regulation by environmental cues. The CREB family of transcription factors participates in a variety of cellular functions including energy homeostasis. Recently, a new family of key CREB coactivators, called TORCs, has been identified. Upon activation, TORC proteins translocate to the nucleus where they potentiate gene activation via a direct interaction with CREB. Drosophila has a single TORC family member, dTORC, which is induced upon fasting, and phosphorylated and degraded upon feeding in an insulin-dependent manner. dTORC mutant flies are sensitive to starvation and have significantly lower glycogen and lipid stores compared to wild type. dTORC functions in the brain, where it is active in a subset of neuroendocrine cells, to regulate the release of an unidentified neuronal signal that instructs the fat body, the energy storage organ, to store glycogen and lipids. This proposal aims to use Drosophila to understand how TORC proteins function to regulate energy balance. Using a rescuing assay of the dTORC mutant phenotype, the specific domains of dTORC required for activation, nuclear translocation and binding to CREB in cell culture assays will be mutated and tested for function in vivo. The hypothesis that dTORC functions in the neuroendocrine cells to produce the signal to the fat body will be tested by supplying dTORC specifically in these cells and assaying for rescue of the mutant phenotype. In vivo nuclear shuttling of dTORC within the neuroendocrine cells in response to feeding and fasting will be examined. From a genetic screen to identify new components of the dTORC pathway, 4 discrete genomic regions have been found to enhance or suppress dTORC function, and thus are excellent candidates for containing genes that encode novel components of the dTORC pathway. The genes within these regions will be identified using single-gene mutations and RNA interference. A screen for genes that affect insulin-dependent dTORC phosphorylation and degradation will be carried out to identify dTORC regulators, leading to an understanding of how insulin signaling regulates dTORC activity. Given the conservation of TORC protein structure and function between Drosophila and mammals, we expect our results to provide key insights into the regulation of energy balance by mammalian TORC proteins.

Project Summary Given its central role in animal health and survival, the gut has evolved a sophisticated network of regulatory inputs from a variety of sources, including immunological, metabolic and microbiotic. Playing a prominent role in this regulatory network is the brain-gut axis, the bi-directional communication between the nervous system and the gut. While there are several well-described brain-gut signals, the exact mechanisms are not always well understood, and many of the signals, particularly stress signals and those involving the microbiome, remain elusive. The fruit fly Drosophila has emerged as a robust model system for unraveling the genetic and cellular basis of human disease, from cancer and neurological disorders to obesity and diabetes. This project seeks to develop Drosophila as a model system to study brain-gut interaction. A novel signal from the fly brain to the gut has been discovered that is activated by metabolic stresses such as fasting. Short Neuropeptide F (sNPF), a relative of mammalian Neuropeptide Y (NPY), is involved in this brain-gut signal. Preliminary studies suggest a model in which sNPF is secreted by a specific set of neurons that integrate the stress signal and innervate the gut. The sNPF signal functions to maintain heightened gut epithelial integrity during periods of stress; in its absence the gut loses epithelial integrity, resulting in an unchecked inflammatory response that depletes energy stores, leading to acute starvation sensitivity and shortened lifespan. The sNPF neurons that innervate the gut will be identified and specifically inactivated using the advanced genetic tools available in Drosophila. If the brain-gut stress signaling model is correct, inactivation of the sNPF neurons should result in the loss of epithelial integrity, inflammatory response and depletion of energy stores. Similarly, activation of the sNPF neurons should lead to enhanced epithelial integrity and resistance to bacterial challenge, and increased energy stores. The model also predicts that sNPF acts directly on the gut via its receptor, sNPF-R. To test this, sNPF-R levels will be knocked down specifically in the gut by RNAi and mutations in the sNPF-R gene will be generated. The discovery and verification of such a brain-gut signal will establish Drosophila as a genetic model system for brain-gut signaling during periods of stress and lay the foundation for identifying novel mechanisms underlying mammalian brain-gut signaling.

Project Summary How neural circuits are assembled during development remains a central unsolved problem in the neurosciences. Finding the genes that control the specificity of synaptic connections within circuits will be one of the keys to eventually manipulating circuit components and promoting functional regeneration following injury. However, elucidating circuit connectivity has been challenging for a number of reasons, including the complexity of the circuits themselves, issues of functional redundancy and the scarcity of genetic tools in many systems to manipulate the development and activity of defined neuronal subsets. This project uses a novel approach that capitalizes on the advanced genetics of Drosophila to define a neural circuit sufficient to drive locomotor behavior, but simple enough that we can determine the relevant synaptic connections and identify the genes controlling its development. Using a temperature-sensitive mutation, shibirets1 that conditionally blocks synaptic transmission at elevated temperatures, neurons within the relatively simple larval nervous system will be synaptically inactivated. By adding back synaptic activity to selected subsets of neurons by targeted expression of wild-type shibire+, a minimal circuit of interneurons, together with sensory and motor neurons, capable of generating motor output will be identified. The long- term goal of this project is to develop Drosophila larval locomotion as a model system for studying circuit development. Given the conservation of nervous system development and function within the animal kingdom, many of the rules by which neural circuits in Drosophila are synaptically connected during development are likely to be directly applicable to understanding the development of mammalian circuits.