Matthew P. Scott - US grants

A basic mystery in developmental biology is how genes function to organize the growth and patterning of the developing embryo. In the fruit fly, Drosophila, genes have been identified which appear to function as developmental switches. A tissue which would normally follow one developmental pathway can be switched to another. Mutations in the genes can lead, for example, to flies with legs developing where the antennae would normally be. A cluster of such development-regulating genes, known as the Antennapedia Complex, has been extensively studied using genetic and developmental approaches. Recently, I have initiated an analysis of the molecular structure and function of the genes in the Complex. The genes have been purified as recombinant DNA. The proposed research is directed at understanding, at the molecular level, how the genes function to control development. We have begun to map genes and transcripts: sixteen mutations have been located on the physical map, and developmental stage-specific transcripts homologous to sections of the cloned DNA have been detected. The specific aims of the project are: 1) to align the physical and genetic maps by precisely mapping more breakpoints of mutations; 2) to locate and characterize each of the transcription units, 3) to identify proteins encoded by the genes, and to localize them in tissues and intracellularly; 4) to analyze the structure, and time and place of expression, of Antennapedia Complex-encoded gene products in mutant flies; 5) to isolate and characterize more mutations in the Complex; and 6) to use the recently developed technique for transforming fly embryos with DNA, and in vitro mutagenesis, to test the function of altered Antennapedia Complex genes in vivo.

A basic mystery in developmental theory biology is how function to organize the growth and patterning of the developing embryo. In the fruit fly, Drosophila, genes have been identified which appear to function as developmental switches. A tissue which would normally follow one developmental pathway can be switched to another. Mutations in the genes can lead, for example, to flies with legs developing where the antennae would normally be. A cluster of such development-regulating genes, known as the Antennapedia Complex, has been extensively studied using genetic and developmental approaches. Recently, I have initiated an analysis of the molecular structure and function of the genes in the Complex. The genes have been purified as recombinant DNA. The proposed research is directed at understanding, at the molecular level, how the genes function to control development. We have begun to map genes and transcripts: sixteen mutations have been located on the physical map, and developmental stage-specific transcripts homologous to sections of the cloned DNA have been detected. The specific aims of the project are: 1) to align the physical and genetic maps by precisely mapping more breakpoints of mutations; 2) to locate and characterize each of the transcription units, 3) to identify proteins encoded by the genes, and to localize them in tissues and intracellularly; 4) to analyze the structure, and time and place of expression, of Antennapedia Complex-encoded gene products in mutant flies; 5) to isolate and characterize more mutations in the Complex; and 6) to use the recently developed technique for transforming fly embryos with DNA, and in vitro mutagenesis, to test the function of altered Antennapedia Complex genes in vivo.

Our long term goal is to understand the molecular mechanisms through which different classes of regulatory genes work together to control development. A fundamental problem in developmental biology is to understand how embryonic cells sense their positions in the embryo and then participate in forming tissues and structures that are appropriate to those positions. This proposal is focused upon genes that appear to be involved in this "positional information" process. In the fruit fly Drosophila extensive gentic screens have led to the identification of many of the genes that control the earliest embryonic pattern formation events such as genes that control the dividion of the embryo into segments or the differentiation of the segments into distinctive structures. Among the segmentation genes is one, patched (ptc), that is likely to be involved in the generation or interpretation of positional information. Mutations in ptc cause embryos to develop with part of the pattern missing from each segment. In each segment, in place of the missing pattern elements is a mirror-image duplication of some of the remaining pattern elements, including the segment boundaries so the mutant embryos have twice the usual number of segment boundaries. No other segmentation gene has a phenotype like that of ptc, although a group of other genes has in common a phenotype involving mirror-image duplications. The ptc phenotype results from incorrect determination of cell fates, and not from death of cells followed by regeneration. Thus cells fail to properly sense their postions, or fail to act appropriately according to their postions. To begin a study of the molecular basis of this phenomenon, DNA clones containing a gene that is likely to be patched have been isolated. We propose a multifaceted study of patched function, including continued developmental analysis, characterization of its interactions with other segmentation genes, investigation of its gene structure, and studies of its protein product(s).

DESCRIPTION (Applicant's Description) Funds are requested for partial support of the 57th Annual Meeting of the Society for Developmental Biology, to be held at Stanford University June 20-25, 1998. We are requesting partial support of the expenses for invited plenary lectures and concurrent symposium speakers, as well as partial support of the travel expenses of minority students. The meeting will have about 800 registrants and will provide opportunities to hear the latest work from international leaders in the field, as well as from younger scientists whose work will be chosen from the abstracts for oral presentation. There will be two large poster sessions, each for two days, allowing everyone a chance to present their work to a richly diverse audience. The topics of the plenary sessions have been chosen to represent some of the most exciting areas of current developmental biology research, including Constructing the Body Plan, Signaling Systems, Neural Development and Patterning, and Cell Guidance Systems. Symposia topics are far ranging: organogenesis, signal transduction, coordination and maintenance of gene regulation, cell biology of development, axial patterning, development and evolution, eye development, medicine and development, morphogenesis, home box gene function and evolution. Work with plants, animals, bacteria, and fungi will be included. Multidisciplinary approached will be emphasized, and special emphasis will be put upon connections with human biology and medicine. Continuing the success of issue-oriented workshops at recent annual SDB meetings, we will hold workshops on education, genomics, applications of computers, funding and future directions in science, ethical issues, and specific research topics such as mesoderm induction. We anticipate a very exciting meeting.

DESCRIPTION (provided by applicant): The normal growth of the cerebellum requires mitogenic signals and feedback mechanisms that limit cell division to the proper times and places. We will investigate mechanisms that control normal cerebellum growth and that fail during cerebellar tumorigenesis. Two growth zones contribute to cerebellar development: the ventricular zone where Purkinje and other cells form, and the external germinal layer that gives rise to granule cells, the most abundant type of neuron in the brain. We have shown that Sonic hedgehog (Shh) signaling protein, produced by Purkinje cells, is a powerful mitogen for cerebellar granule cell precursors. The Patched 1 transmembrane protein, produced in granule cell precursors, is a receptor for Shh. Shh activates the transcription of target genes by preventing Patched1 from inhibiting their transcription. We have shown that reduced human PATCHED1 function is associated with sporadic and inherited types of medulloblastoma. Medulloblastoma is the most common type of childhood malignant brain tumor, and it has an approximately 50 percent mortality rate. We have made a mouse model of medulloblastoma based on the human genetics of the disease, with reduced patched1 function leading to frequent tumors. The tumor cells are marked by lacZ expression, so they can be recognized long before an overt tumor forms. This allows investigation of early stages of tumorigenesis. We propose experiments to learn how Shh signaling controls normal granule cell development by affecting cell cycle and other regulators, and how reduced Patched 1 function leads to medulloblastoma. The specific aims are: 1. To investigate Shh regulation of the cell cycle and cell differentiation in cerebellar granule cell precursors. 2. To investigate how granule cell precursors stop responding to mitogenic effects of Shh and begin differentiation and migration. 3. To use mouse models of medulloblastoma to investigate mechanisms of tumorigenesis.

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is employed in controlling cell fates in most developing tissues and organs, as well as during many regeneration events. Defects in Hh signaling lead to birth defects and cancer. Many mechanistic mysteries remain regarding how an Hh signal is transduced. Using high-throughput RNAi screening, we identified Neuropilins (Nrp) 1 and 2 as novel, specific regulators of vertebrate Hh signaling. Nrps are single-pass transmembrane proteins implicated in the reception of a diverse set of secreted ligands, including Semaphorins and VEGF165 and in cell adhesion and cell migration. In fibroblasts the inhibition of Hh signal transduction resulting from blocking Nrps is as strong as the effect of blocking cilia formation or blocking Smoothened function. Conversely, over-production of either Nrp sensitizes cells to Hh signals. New components of the Hh pathway are uncovered infrequently; the Nrps were probably missed due to their partial redundancy. Our discovery of two proteins whose functions are required by this important morphogenic pathway has the potential to bring fundamental changes to current models of Hh signaling and to enlarge the understanding of Nrp functions in other signaling pathways. Aim 1. Determine the mechanism by which Nrps regulate Hh signal transduction. Nrps could influence Hh signal transduction by directly associating with known Hh pathway components, or mediating other signals that converge with Hh transduction, or by altering cell properties or processes that are required for Hh transduction. We will investigate each of these possibilities by determining which steps in Hh signaling are affected, whether cell adhesion or migration changes are involved in the effect of Nrps upon Hh signaling, and what proteins interact directly with Nrps. Aim 2. Determine which domains of Nrp are needed to support Hh signaling, and whether known Nrp co-receptors, ligands, or effector molecules are capable of Hh pathway cross- regulation. We will investigate which domains contribute to Hh signal transduction in two ways: engineered domain deletions, and a high-throughput screen for point mutations that interfere with Nrp support of Hh signal transduction. Nrps transduce VEGF and Semaphorin signals, acting as co-receptors for VEGF receptors and Plexins, respectively. We will test whether VEGF, VEGF receptor, Plexin receptors, or Semaphorins are involved in the effect of Nrps upon Hh signal transduction. Aim 3. Investigate how Nrps influence Hh- dependent development and tumorigenesis. Using mice that carry mutations in both Nrp genes, we will control temporal and tissue-specific removal of Nrp functions to test their involvement in several Hh-dependent developmental processes in vivo. Using newly created lentiviruses, which encode specific inhibiting RNAs that block the nrp genes, we will infect primary cultures of Hh-responsive cells and monitor effects on Hh target gene expression.

DESCRIPTION (provided by applicant): Hedgehog (Hh) signaling is fundamental to the control of differentiation and growth. During development of the cerebellum, Purkinje neurons emit Sonic hedgehog (Shh), a potent mitogen for adjacent granule neuron precursors (GNPs). GNPs respond to Shh by altering the processing, location, and modification of Gli transcription factors that activate or repress target genes. Mutations in human or mouse patched1, which encodes the Shh receptor, promote transformation of GNP cells into medulloblastomas (MBs), the most common childhood malignant brain tumor. We used chromatin immunoprecipitation (ChIP) and high-throughput sequencing to identify locations of Gli1 binding in the chromatin of murine GNPs and MB cells. This led us to Gli-responsive transcriptional enhancers. Combining ChIP data with gene expression data we identified putative target genes that are directly regulated by Shh. Dramatic changes in targeting of Gli1, and target gene expression, occur when cerebellum precursor cells become cancer cells. We will investigate the mechanisms of target gene selection, the connections between Hh target genes and the cell cycle, and the roles of target genes in normal development and tumorigenesis. Specific Aim 1: Investigate how Gli transcription factors coordinate to regulate gene expression in cerebellum development and tumorigenesis. Our ChIP data led to many novel target genes, and well-established targets like Ptch1, Gli1, and N-myc. 132 genes are consistent targets in normal and tumor cell types. Remarkably, despite the close relation between GNPs and MB cells, many putative target genes are specific to one cell type or the other. We will determine the mechanism of selective recognition of enhancer elements in the two cell types. Specific Aim 2: Determine how chromatin modifiers influence Gli-regulated gene expression. We have identified histone modifications that correlate with the regulatory changes for specific target genes in GNPs vs. MBs. We will investigate the mechanistic importance of these changes in Hh target gene specification. Specific Aim 3: Investigate interactions of Gli proteins with other transcription factors. Computational analyses of the DNA regions bound by Gli1 revealed evidence for two types of transcription factors, E box-binding proteins and NFI proteins, that may work in parallel, or collaborate, with Gli1 protein. We will investigate their roles in target gene control. Specific Aim 4: Define contributions of Gli targets to GNP development and tumorigenesis. We will investigate selected target genes that mediate the connection between Hh signaling and the cell cycle, in the context of GNPs and tumors. The planned studies have direct importance for understanding developmental and tumorigenic roles of the Hedgehog pathway in many tissues and organs. Discovering genes that are directly regulated by Hh signaling will lead to new ways to intervene when errors in signal transduction lead to birth defects or cancer.

DESCRIPTION (provided by applicant): Niemann Pick type C (NPC) disease is a fatal pediatric disorder. The disease is due to mutations in either of two genes, NPC1, which encodes a 13 transmembrane domain sterol-binding protein, and NPC2, which encodes a soluble sterol-binding protein. Loss of either gene causes aberrant organelle trafficking and accumulation of free cholesterol within lysosomes. NPC patients suffer from hepatomegaly and progressive cognitive and locomotion losses, with massive Purkinje neuron (PN) death in the cerebellum. There is no effective treatment for NPC. Specific Aim 1: Determine why Purkinje neurons die in NPC disease and at what stages the disease can be arrested or reversed. We will provide a functional tagged- Npc1 protein to specific classes of cells, eg neurons, astrocyte, or liver cells, in the Npc1-/- background, to define how each cell type contributes to disease. Our transgenes are engineered with Tet-technology to allow cell-specific and temporal regulation of tagged Npc1 production. Specific Aim 2: Learn how NPC disease affects intracellular trafficking in Purkinje neurons. Loss of Npc1 causes striking intracellular trafficking defects in fibroblasts. To learn how PNs are affected, we will culture them and track the trafficking of NGF and other molecules labeled with quantum dots. We will use a portable two-photon microscope in combination with organelle dyes to characterize vesicular movements in living astrocytes and PNs of wild-type and Npc1-/- mice. In living brains of our newly engineered mice we will analyze movements of Npc1-positive organelles in PNs, other neurons, and astrocytes to determine what changes may cause cell death. Specific Aim 3: Determine whether inflammation protects from, or causes, NPC cell death. We will produce functional Npc1 in neurons, hepatocytes, and inflammatory cells, in otherwise Npc-/- mice, and see which of these is most effective in preventing inflammation. We will use mouse mutations that reduce inflammation in combination with Npc1-/-, and see whether PN survival and liver pathology are improved or worsened. We will ablate hepatic macrophages with chlodronate-filled liposomes and assess the role of macrophages in NPC liver damage. Specific Aim 4: Discover whether autophagy protects from, or causes, NPC cell death. We will cross NPC mice with mutants that cannot trigger autophagy: Toll like receptor-7 and beclin-1 deficient mice. Conversely we will test mice that have over-expressing Beclin1 in neurons and consequent have heightened autophagy, or enhance autophagy with Beclin1 virus infections or rapamycin treatments. Lysosome storage disorders like NPC encompass nearly 60 different conditions, most of which damage liver and/or brain function. Some, including NPC, have similarities to Alzheimer disease. We have used a novel approach to engineer mice that will allow us to learn how different cell types and processes contribute to disease. Learning the roles of inflammation and autophagy in NPC neurodegeneration has direct implications for therapeutic interventions that will arrest or reverse disease progression. PUBLIC HEALTH RELEVANCE: Niemann-Pick C syndrome is a neurodegenerative disorder involving failures of intracellular transport of organelles, and accumulation of large quantities of cholesterol, and these problems lead to liver and brain damage. We have engineered mice in which specific types of cells can be rescued from the disease while other cells remain mutant. We will use these mice to learn how different types of cells contribute to NPC disease pathology, and to investigate how inflammation, and attempts by cells to survive by consuming their own proteins, are involved in the deaths of neurons and liver cells.

DESCRIPTION (provided by applicant): The development of most organs and tissues requires signal transduction pathways such as Hedgehog (Hh), TGF1, Notch, and Wnt. Most studies have focused on established components of these pathways, information flow between them, and the relatively slow transcriptional changes they drive. We propose instead a new systematic approach to discovering important rapid responses to developmental signals, using Hh signaling as a test case. Specific Aim 1: We will identify immediate-early responses to an important developmental signal, Hedgehog, applying Stable Isotope Labeling with Amino acids in Cell culture (SILAC) and mass spectrometry to developmental regulation. Specific Aim 2: We will delineate the functions of re-discovered components in the Hh immediate-early response and explore novel leads that will provide insight into signal transduction. Summary: Comprehensive assessment of the earliest responses to major developmental signals will reveal new and critical aspects of the signal transduction mechanisms. Our tests using one pathway will set the stage for applying the approach to many others.

Cells; Systems Biology;