David J Anderson
California Institute Of Technology
Project start date: 2009-06-15
Project end date: 2014-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to David J Anderson
GENETIC ANALYSIS OF EPHRIN-EPH SIGNALING IN ANGIOGENESIS
David J Anderson, Roger W. Sperry Professor
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5R01HL066221-05 from National Heart, Lung, And Blood Institute IRG: ECS
Abstract: Verbatim ) The transmembrane ligand ephrinB2 is specifically expressed by arterial but not venous endothelial cells in both embryos and adults, whereas its receptor EphB4 is conversely expressed by veins but not arteries. Both the ligand and receptor are essential for embryonic cardiovascular development. The objective of this proposal is to understand more precisely the function of ephrinB2-EphB4 signaling in angiogenesis. Using conditional loss-and gain-of-function manipulations in knockout and transgenic mice, we will test the hypothesis that bi-directional signaling between arteries and veins mediated by this ligand-receptor pair is essential for angiogenesis, and ask whether the cellular function of this signaling is primarily attractive or repulsive. In Specific Aim I, we will knock out ephrinB2 specifically within endothelial and endocardial cells, to determine whether its essential function is indeed exerted within the circulatory system. These studies will be complemented by experiments to selectively rescue the ephrinB2 knockout phenotype within the circulatory system. Temporally controlled, pan-endothelial knockout of ephrinB2 will also be performed, to determine whether the gene is also required at later stages of angiogenesis. In Specific Aim II, we will use both in vitro embryoid body assays of blood vessel formation and in vivo embryo chimeras in conjunction with heterozygous and homozygous ES cells mutant for ephrinB2 or EphB4 to distinguish whether the primary requirement for these genes in early cardiovascular development is in the heart, the peripheral vasculature, or both. In Specific Aim III we will perform constitutive and/or conditional pan-endothelial expression of ephrinB2 and EphB4 transgenes to determine whether the arterial- and venous-specific expression, respectively, of these genes is essential for their proper function in the circulatory system. In Specific Aim IV we will create lines of "dual-indicator" mice that can be used to simultaneously distinguish arteries and veins using genetically encoded photochemical markers. These mice will be used in both in vivo and in vitro experiments to further study the role of cell-cell interactions between arteries and veins and the role of ephrinB2-EphB4 signaling in mediating these interactions. Mechanistic studies of the role of ephrinB2-EphB4 signaling in development are highly like to inform our understanding of its function in adult angiogenesis, and may suggest new therapeutic strategies for the inhibition or promotion of angiogenesis in clinical settings such as cancer and heart disease, via pharmacological manipulation of these artery- and vein-specific signaling molecules.
Keywords: angiogenesis, artery, biological signal transduction, ephrin, intermolecular interaction, ligand, membrane protein, receptor, capillary bed, cell cell interaction, endocardium, gene deletion mutation, gene expression, gene targeting, genetic manipulation, muscle cell, peripheral blood vessel, protein structure function, smooth muscle, tissue mosaicism, vascular endothelium, vein, animal breeding, cell line, embryo /fetus transplantation, genetically modified animal, laboratory mouse
Project start date: 2000-12-18
Project end date: 2007-11-30
5R01HL066221-05 (2005): $380573
5R01HL066221-04 (2004): $380993
5R01HL066221-03 (2003): $381401
5R01HL066221-02 (2002): $342297
1R01HL066221-01 (2001): $342682
PREDOCTORAL TRAINING IN BIOLOGY AND BIOPHYSICS
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5T32GM007737-25 from National Institute Of General Medical Sciences IRG: BRT
Abstract: This program will provide predoctoral training of students preparing for research careers in Molecular, Cellular, and Systems Neuroscience. It involves 26 faculty members from the Biology, Physics, Chemistry, and Engineering Divisions. It is a continuation of a program previously supported by NIH. Some research areas of special emphasis are 1) neural development (control of cell fate, axon guidance and synaptogenesis in a variety of systems); 2) signal transduction mechanisms in neurons (sensory processing in the visual and olfactory systems of vertebrates and invertebrates, and synaptic transmission and plasticity in hippocampal neurons); 3) behavior (simple and complex behaviors in vertebrates, arthropods, and nematodes); 4) computational neuroscience (studies of single neurons, system of neurons, and whole organisms). The major components of our training activities are 1) each student s individual research program under one or more faculty sponsors; 2) an organized curriculum of graduate courses; 3) preparation for qualifying examinations; 4) teaching activities; 5) an extensive and wide-ranging seminar program. Support is requested for 16 predoctoral trainees, who will be admitted to graduate study for a Ph.D. in Biology or in Computation and Neural Systems. Criteria for admission into the program include a strong motivation for a career in research and high quantitative ability. Our expectation that trainees will continue into productive research careers is supported by the records of previous trainees. Caltech has a strong commitment (at both the institutional and the Divisional levels) to increasing the representation of minorities in science. In the Biology program, we have made special efforts to attract exceptionally talented students from under-represented minority groups, and we have been quite successful in this effort in recent years. A number of these students are primarily interested in neuroscience research. The training faculty are located within several building clustered near each other on the Caltech campus. Multi-user facilities include DNA sequencing, oligonucleotide synthesis, peptide synthesis, protein expression and purification, monoclonal antibody production, a transgenic and knockout mouse facility, and the Biological Imaging facility.
Project start date: 1979-07-01
Project end date: 2004-06-30
5T32GM007737-25 (2003): $392815
5T32GM007737-24 (2002): $414353
5T32GM007737-23 (2001): $392304
PREDOCTORAL TRAINING IN INTEGRATIVE NEUROSCIENCE
David J Anderson, Professor
California Institute Of Technology, Office Of Sponsored Research, Mail Code 201-15, Pasadena, Ca 91125
Grant 3T32GM007737-30S1 from National Institute Of General Medical Sciences
Abstract: This program will provide predoctoral training of students preparing for research careers in Molecular, Cellular, and Systems Neuroscience. It involves 25 faculty members from the Biology, Physics, Chemistry, and Engineering Divisions. It is a continuation of a program previously supported by NIH. Some research areas of special emphasis are 1) neural development (control of cell fate, axon guidance, and synaptogenesis in a variety of systems); 2) signal transduction mechanisms in neurons (sensory processing in the visual, auditory, somatosensory and olfactory systems of vertebrates and invertebrates, and synaptic transmission and plasticity in hippocampal neurons); 3) behavior (simple and complex behaviors in vertebrates, arthropods, and nematodes, including behavioral genetics); 4) computational neuroscience (studies of single neurons, systems of neurons, and whole organisms). The major components of our training activities are 1) each student´s individual research program under one or more faculty sponsors; 2) a required course and an organized curriculum of elective graduate courses; 3) preparation for qualifying examinations; 4) teaching activities; 5) an extensive and wide-ranging seminar program; 6) regular presentations by students on their research progress; 7) a Neuroscience Retreat designed to foster intellectual crossfertilization among trainees. Support is requested for 16 predoctoral trainees, who will be admitted to graduate study for a Ph.D. in Biology or in Computation and Neural Systems. Criteria for admission into the program include a strong motivation for a career in research and high quantitative ability. Our expectation that trainees will continue into productive research careers is supported by the records of previous trainees. Caltech has a strong commitment (at the Training Program, Divisional and Institute levels) to increasing the representation of minorities in science. In the Biology program, we have made special efforts to attract exceptionally talented students from underrepresented minority groups, and have been quite successful in this effort in recent years. A number of these students are primarily interested in neuroscience research. The training faculty members are located within several buildings clustered near each other on the Caltech campus, including the newly-completed Broad Center for the Biological Sciences. Multi-user facilities include the Biological Imaging facility, a transgenic and ´knockout´ mouse facility, a new fMRI facility located in the Broad Building, and facilities for DNA sequencing, peptide synthesis, protein expression and purification, monoclonal antibody production, electron microscopy and flow cytometry
Project start date: 1979-07-01
Project end date: 2010-06-30
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
3T32GM007737-30S1 (2009): $189550
5T32GM007737-30 (2008): $0
Sponsored Links Excellgen http://Excellgen.com
5T32GM007737-29 (2007): $345174
5T32GM007737-28 (2006): $406748
5T32GM007737-27 (2005): $447422
2T32GM007737-26 (2004): $447422
MOLECULAR AND CELLULAR CONTROL OF SENSORY NEUROGENESIS
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5P50MH049176-080011 from National Institute Of Mental Health
Abstract: The overall goal of this project is to understand the mechanisms that generate different classes of neurons during development. We are using the neural crest, which generates the peripheral nervous system, as a model system. Our previous work has focused on the mechanisms that generate autonomic neurons. In this proposal we shall investigate the control of sensory neurogenesis, which has been relatively understudied due to lack of adequate markers. We will carry out these studies at both the cellular and molecular level. Specifically, we will generate and analyze mice containing targeted mutations in neurogenin-1 (ngn-1) and neurogenin-2 (ngn-2), two genes encoding NeuroD-related basic-helix-loop-helix transcriptional regulators that are specifically expressed early in sensory gangliogenesis, as well as in the basal layer of the olfactory epithelium. Using a recently-developed primary explant system for sensory neurogenesis, we will examine the regulation and function of neurogenin-1 and neurogenin-2 in vitro, to complement the in vivo analysis of mutant mice. We will also use this explant system as an assay for putative sensory neuron progenitors, that we propose to isolate by fluorescence-activated cell sorting from heterozygous mice in which the green lantern protein (GLP) has been inserted into the ngn-1 locus by homologous recombination. These isolated sensory neuron progenitors will be immortalized using a recently-developed conditional system, to generate cell lines that can provide large numbers of cells for further study of sensory neuron development and function. In parallel, a similar approach will be undertaken to isolate and immortalize olfactory neuron stem cells from the olfactory epithelium, based on their expression of GLP in the ngn-I locus. Studies of olfactory receptor expression and function in these cell lines will be carried out in collaboration with K. Zinn.
Keywords: gene expression, neural crest, neurogenesis, neuron, olfactory lobe, cell differentiation, developmental genetics, epithelium, receptor expression, transcription factor, autoradiography, cell line, flow cytometry, gene targeting, green fluorescent protein, in situ hybridization, laboratory mouse, laboratory rat, restriction endonuclease, tissue /cell culture
Molecular Control Of Neural Cell Fate Determination
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 2R01NS023476-19 from National Institute Of Neurological Disorders And Stroke IRG: NCF
Abstract: The objective of this proposal is to understand the cellular and molecular mechanisms that control a fundamental event in neural development the switch from neurogenesis to gliogenesis. An understanding of this process is essential for applying neural stern and progenitor cell biology to the treatment of neurological disease. This switch will be studied in a specific population of spinal cord precursors that sequentially generate motoneurons (MNs) and oligodendracytes (oligos). These precursors can be prospectively isolated using fluorescence-activated cell sorting (FACS), by means of a GFP reporter expressed from the Olig2 locus, which encodes a transcription factor required for both MN and oligo differentiation. Using these isolated cells, we will address the following specific aims I). We will test whether MNs and oligos develop from a multipotential, self-renewing stem cell in the ventricular zone (VZ) of the spinal cord, as is widely assumed, or rather from progenitors that undergo irreversible restrictions in developmental competence. We will investigate this by using a newly developed technique for direct transplantation of freshly isolated Olig2-expressing progenitors into the chick spinal cord, without any ex vivo expansion (which may perturb the properties of the cells). Using this approach, we will perform heterochronic transplantation experiments to test the self-renewal anc developmental capacities of Olig2-i- cells at different stages during the MNgoligo transition. II). We will test the hypothesis that changes in gene expression in Olig2+ progenitors during the neuron-to-glial switch reflect the regulation of several distinct subclasses of genes, each with different kinetics of activation and repression. This hypothesis will be tested by using oligonucleotide microarrays to perform gene expression profiling (GEP) experiments on acutely isolated Olig2+ progenitors from different stages of spinal cord development. This GEP analysis should also identify a) markers useful in clarifying lineage relationships between Olig2+ progenitors of MNs and oligos; and b) candidate regulatory genes for functional analysis. Ill) We will tost the hypothesis that there is a "temporal code" of transcription factors that controls the MN->oligo switch, by performing gain-of-function (GOF) and loss-of-function (LOF) genetic manipulations of candidate regulatory genes identified in the GEP temporal analysis (Aim II). Electroporation of chick spinal cord will be used as a rapid in vivo assay for such functional manipulations, employing expression of full-length cDNAs, and independently validated shRNAi (small hairpin RNAi) constructs, for GOF and LOF experiments, respectively. The embryos will be analyzed uning an extensive battery of molecular markers for various classes of neurons (including MNs and interneurons), oligodendrocytes, and newly validated markers of astrocytes and their progenitors. Candidates for which strong functional data is obtained from the chick system will be further validated by generating constitutive or conditional knockouts in the mouse. IV) We will test the hypothesis that targets of OLIG2, which functions as a transcriptional repressor, include a) repressers of oligo differentiation; and b) activators of astrocyte differentiation. Candidate targets of OLIG2 will be identified by performing comparative GEP analysis of isolated Olig2-GFP+ cells from Olig1/2+/- and Olig2-/- spinal cord, at several developmental stages. A series of analytic algorithms will be used to filter the data to obtain a list of transcription factors that are de-repressed in the absence of OLIG2 function. These candidates will be further validated and prioritized by real-time RT-PCR and in situ hybridization. Top candidates will then be functionally analyzed by GOF and LOF manipulations in chick embryos.
Keywords: cell differentiation, developmental genetics, developmental neurobiology, gene expression, genetic regulation, motor neuron, neurogenesis, oligodendroglia, cell cell interaction, cell population study, gene interaction, genetic mapping, nerve stem cell, protein structure function, spinal cord, transcription factor, cell transplantation, chick embryo, flow cytometry, gene expression profiling, genetically modified animal, in situ hybridization, laboratory mouse, polymerase chain reaction, tissue /cell culture
Project start date: 1986-09-01
Project end date: 2010-04-30
2R01NS023476-19 (2005): $360804
Region-specific, Inducible Axonal Tract-tracing In Brain
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5R01MH070053-04 from National Institute Of Mental Health IRG: ZRG1
Abstract: The objective of this proposal is to develop improved methods for inducible genetic marking, mapping and manipulation of specific brain regions or neuronal subpopulations in the mammalian brain. In Aim I, methods for isolating brain region-restricted and cell type-specific genes utilizing laser-capture microdissection together with DNA microarray analysis will be developed and optimized. These experiments will focus on identifying markers for limbic system structures, including the amygdala, BNST, hypothalamus and lateral septum. In Aim II, the utility and efficacy of different genetically encoded primary axonal markers will be compared, and genetically encoded, inducible anterograde and retrograde trans-neuronal tracers will be developed and tested in vivo. Proof-of-principle will first be established in the PNS using a well-defined model system, and then extended to the CNS. Aim III encompasses the development and in vivo testing of two alternative combinatorial, positive coincidence-detection systems for achieving region- or cell subtype-specific control of heterologous gene expression (e.g., neuronal tracers or silencers), without having to identify specific transcriptional enhancer elements for such regions or cells. One method is based on inducible site-specific DNA recombination. The other method is based on a "two-hybrid" system for inducible transcriptional activation. In both cases, expression of the reporter/tracer gene is induced only in cell populations in which the expression of two different "co-driver" genes overlaps. This "Venn diagram" approach allows different pairwise combinations of driver genes to be used to express reporter or tracer genes in only a restricted subset of the regions in which each individual co-driver gene is expressed. These methods will initially be developed, tested and optimized using highly specific genes with known overlapping patterns of expression in specific subsets of pain-sensing primary sensory neurons. In Aim IV, based on the outcome of Aim III, either the recombination-based or two-hybrid system will be selected for extension to the CNS, using an overlapping pair(s) of limbic system-specific genes identified in Aim I. In addition, the recombination-based system will be extended to permit activity-dependent trans-neuronal tracing of neurons expressing a specific marker gene. The combination of new limbic system-specific molecular markers and genetically encoded tract-tracing and neuronal ablation methods should improve our understanding of the functional neuroanatomy of emotion and affective disorders such as anxiety and depression.
Keywords: brain mapping, gene expression, genetic marker, method development, axon, limbic system, neuron, confocal scanning microscopy, dissection, genetically modified animal, in situ hybridization, laboratory mouse, microarray technology
Project start date: 2004-01-01
Project end date: 2008-12-31
5R01MH070053-04 (2007): $304755
5R01MH070053-03 (2006): $313856
Sponsored Links Excellgen http://Excellgen.com
5R01MH070053-02 (2005): $321410
1R01MH070053-01 (2004): $321410
David J Anderson
California Institute Of Technology, Office Of Sponsored Research, Mail Code 201-15, Pasadena, Ca 91125
Abstract: The Mouse Core plays a central role in this proposal, providing for the breeding and management of the large colony of transgenic and knockout mice that will be generated for this project. Five different knockout strains have been or will be created for this project, and these will be each intercrossed with one another, as well as with 3-4 different strains of transgenic lines (tetO-responder lines), to generate multiple substrains. These animals are the source for all of the behavioral, physiological, pharmacological, anatomical and cell-based studies in the Program Project, and are a primary shared resource for all of the collaborative experiments among the three participating laboratories. The need for this core is justified by the large numbers of genetically modified mice that must be generated and maintained solely for the purposes of this project (see Budget Justification). The behavioral phenotyping experiments in particular require large numbers of animals of each strain and genotype, because the greater inherent variability of such experiments requires large numbers of animals to achieve statistical significance. The Mouse Core will leverage the infrastructure, personnel, equipment and other resources available in the Transgenic Animal Facility at Caltech (TAFCIT), while providing exclusive support for the generation, maintenance, breeding and shipping of transgenic and knockout mice strains associated with this Program Project
Keywords: Animal Sources; Animals; Behavioral; Breeding; Budgets; Cells; Equipment; Family; Generations; Genotype; Human Resources; Infrastructure; Knock-out; Knockout; Knockout Mice; Laboratories; Maintenance; Maintenances; Mammals, Mice; Manpower; Mice; Mice, Knock-out; Mice, Knockout; Mouse Strains; Murine; Mus; Nociception; Null Mouse; Phenotype; Physiologic; Physiological; Play; Programs (PT); Programs [Publication Type]; Research Infrastructure; Research Resources; Resource Sharing; Resources; Role; Shipping; Ships; Transgenic Animals; Transgenic Organisms; animal facility; base; experiment; experimental research; experimental study; nociceptive; personnel; programs; research study; social role; transgenic
Budget start date: 1-SEP-2009
Budget end date: 31-AUG-2010
3P01NS048499-05S1_9001 (2009): $15724
3P01NS048499-05S2_9001 (2009): $14116
Molecular Control Of Neural Cell Fate Determination
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5R01NS023476-18 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The long-term goal of this proposal is to understand the cellular and molecular mechanisms that control cell fate determination in the developing vertebrate nervous system, with a particular emphasis on the choice between neuronal and glial fates. Specifically, we propose to study the function and regulation of Olig1 and Olig2, two basic helix-loop-helix (bHLH) transcription factor genes we have recently isolated that are specifically expressed in the oligodendrocyte lineage (Zhou et al. (2000) Neuron 25331). In Specific Aim I of this proposal, we will determine whether the Olig genes are essential determinants of the oligodendrocyte fate in vivo, by generating Olig1 and 2-deficient mice and analyzing the phenotypes of various allelic combinations of these mutations, using an extensive battery of molecular markers. In Specific Aim II, we will determine whether expression of Olig genes marks commitment to the oligodendrocyte lineage, by using vital markers incorporated into the gene targeting cassettes to isolate Olig1- and Olig2-expressing cells by flow cytometry. The developmental capacities of these isolated cell populations will be tested by in vivo transplantation and by in vitro cell culture. In the second part of this proposal we will investigate the regulation of Olig gene expression and its functional interactions with other determinants of neural cell fate in the spinal cord. The expression of Olig genes defines a restricted zone from which oligodendrocyte precursors will emerge several days later. In Specific Aim III, we will carefully map the relationship of the Olig gene expression domain to the domains of expression of other known regulators of spinal cord cell fate, using double- and triple-labeling with antibodies and/or cDNA probes and laser scanning confocal fluorescence microscopy. These latter regulators include components of a recently identified homeodomain code that specifies different progenitor domains (Briscoe et al. (2000) Cell 101435); neurogenic bHLH factors and Notch ligands. In Aim IV, we will functionally test hypotheses for the regulation of Olig gene expression suggested by correlative data obtained in Aim III, using retrovirus- or electroporation-mediated gene transfer to mis-express candidate regulatory genes in the chick spinal cord and determine their effect on the domain of Olig gene expression, and vice-versa. An understanding of the cellular and molecular mechanisms that control oligodendrocyte lineage determination in neural progenitor cells is an essential prerequisite to the controlled manipulation of such cells for transplantation therapy of neurological diseases such as Multiple Sclerosis.
Keywords: cell differentiation, developmental genetics, developmental neurobiology, gene expression, genetic regulation, glia, neurogenesis, neurogenetics, neuron, transcription factor, DNA binding protein, astrocyte, cell population study, genetic mapping, oligodendroglia, protein structure function, spinal cord, cell transplantation, chick embryo, confocal scanning microscopy, electroporation, gene targeting, genetically modified animal, laboratory mouse, tissue /cell culture, transfection /expression vector
Project start date: 1986-09-01
Project end date: 2005-04-30
5R01NS023476-18 (2004): $304690
5R01NS023476-17 (2003): $304690
Sponsored Links Excellgen http://Excellgen.com
5R01NS023476-16 (2002): $304690
2R01NS023476-15 (2001): $304690
MOLECULAR BIOLOGY OF NEURAL CREST DEVELOPMENT
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5R01NS023476-14 from National Institute Of Neurological Disorders And Stroke IRG: NEUC
Abstract: The long-range goal of this project is to understand the mechanisms controlling the lineage commitment and differentiation of multipotent neural progenitor cells, using the neural crest as a model system. The focus of this proposal is to understand the function and regulation of NRSF, a transcriptional repressor of neuronal genes previously identified in this laboratory. NRSF is expressed in most non-neuronal tissues, as well as in undifferentiated neuroepithelium; however it is not expressed in most neurons. This suggests that NRSF may silence neuronal genes in non-neuronal cells, and/or may transiently repress such genes in neural progenitor cells prior to neuronal differentiation. The latter function would in turn require a mechanism to repress NRSF during neurogenesis. Several parallel and complementary approaches will be undertaken to address these questions. Structure-function analysis, using in vitro DNA binding assays and in vivo silencing assays in transfected mammalian cells, will dissect domains of NRSF important in repression. Further light can be shed on the mechanism of repression by identifying other proteins with which these domains interact, using the yeast 2-hybrid system screen. NRSF domains will also be used to design dominant-negative forms that can be used to perturb NRSF function in various experimental systems, including cultured neural crest cells and Xenopus embryos. Targeted mutations in the NRSF gene will be generated in mice and the phenotype of these mutants characterized using a variety of molecular markers, both in vivo and in cultures of neural crest cells established using a variety of molecular markers, both in vivo and in cultures of neural crest cells established from mutant embryos. The regulation of environmental factors will be examined in primary cultures of neural crest cells grown under conditions that promote commitment to different lineages. The interaction of NRSF with the regulatory circuits that control neuronal differentiation in neural crest cells will be investigated by constitutively expressing intact or mutant NRSF from retroviral vectors in these cells, and examining the response to factors such as BMP2 that promote neurogenesis as well as the expression of positive transcriptional regulators of neurogenesis such as MASH-1. An understanding of the molecular mechanisms that control the commitment and differentiation of multipotential neural progenitors to different lineages will be valuable for human health, both in developing potential cell-replacement therapies for neurologic and neurodegenerative diseases and for developing anti-tumor therapies for cancers of the neural crest such as neuroblastoma.
Keywords: developmental genetics, developmental neurobiology, neural crest, neurogenesis, neurogenetics, pluripotent stem cell, gene induction /repression, gene mutation, invertebrate embryology, mammalian embryology, neuron, protein structure /function, transcription factor, Xenopus, Xenopus oocyte, animal genetic material tag, laboratory mouse, tissue /cell culture, transgenic animal
Project start date: 1986-09-01
Project end date: 2001-04-30
5R01NS023476-14 (2000): $189891
5R01NS023476-13 (1999): $184361
5R01NS023476-12 (1998): $178994
2R01NS023476-11 (1997): $173781
5R01NS023476-09 (1994): $314589
5R01NS023476-08 (1993): $300037
5R01NS023476-07 (1992): $288500
Sponsored Links Excellgen http://Excellgen.com
THE GORDON CONFERENCE ON NEURAL DEVELOPMENT
David J Anderson
Gordon Research Conferences
west Kingston, Ri 02892
Grant 1R13NS040252-01 from National Institute Of Neurological Disorders And Stroke IRG: NSD
Abstract: The Gordon Conference on Neural Development has evolved into one of the key meetings in the field. The subject of developmental neurobiology is very complex, requiring integration of hypotheses and information at the molecular, cellular, and systems levels. The conference is intended to bring together such a mixture of research groups in a format highly conducive to both formal and informal exchange. The emphasis will be on the discussion of cutting-edge, unpublished research. In addition, the breadth of the meeting provides an excellent opportunity for those who are beginning their careers or moving into a new subject area. The meeting is small (approximately 125 participants); however, the Chair and Vice-Chair will strive to ensure that a diverse group of both junior and senior investigators attends. The financial support requested will also be used to increase the numbers of women and minorities participating in this meeting. The speakers we have chosen represent not only some of the most active groups, but also individuals with the capacity to generate useful discussion of their own and other topics. The concept of the meeting has been not to try to cover the entire field thinly, but to focus on areas of exceptional activity or promise. Featured in this year´s program are the following topics activity vs. genetic specification in the formation of neuronal connections, identity of stem cells in the adult brain, control of the neuron/glia fate choice, neuronal polarity, expression and functions of neuronal cadherins, axon guidance, transcriptional codes in neuronal identity, and development of sensory systems. Within these topics, we have included several speakers whose research has important clinical applications. The meeting is well-balanced, containing both promising young investigators as well as more senior leaders in the field. Forty-five to fifty minutes will be allowed for each speaker´s topic, of which one- third will be devoted to discussion. The afternoons are open for informal interactions. Several poster sessions in which conferees can present their work will be scheduled; these have been extremely well attended at previous conferences. Most participants will be expected to present a poster, thus this meeting will serve both a scientific and training function
Keywords: developmental neurobiology, meeting /conference /symposium, neurogenesis brain cell, cadherin, developmental genetics, neurogenetics, neuronal guidance, stem cell travel
Project start date: 2000-06-01
Project end date: 2001-05-31
1R13NS040252-01 (2000): $25000
MOLECULAR FATE MAPPING IN MOUSE USING CRE RECOMBINASE
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5P01AR042671-050002 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases
Abstract: The goal of this project is to develop a new molecular method, termed recombinase-based lineage tracing" (RBLT), to provide a fate map of embryonic precursor cells that express the MASH-1 gene, a nuclear regulatory gene of the basic helix-loop-helix (HLH) family. Recombinase mediated lineage tracing provides a means to mark the mitotic progeny by DNA rearrangement driven by a site-specific recombinase (Cre). A primary advantage of this approach is the access to neural precursor cell populations that would likely be inaccessible to dye labeling or retroviral marking methods. Since the review of the original application, several significant advances have been made. First, the investigators demonstrated that Cre-mediated recombinations of a lacZ reporter gene are feasible for fate mapping experiments, shown by crossing activator mice expressing a CMV-Cre construct to transgenic animals harboring a reporter gene, chick actin XSTOPX lacZ. The offspring generated showed no developmental defects, thus offsetting a concern raised in the previous review. Second, the investigators will utilize the green fluorescent protein (GFP) as an alternative to alpha-galactosidase. Finally, a recent publication describing a null mutation in the MASH-1 gene demonstrated an essential role for MASH-1 in the development of several classes of neurons, including the sympathoadrenal lineage and olfactory neurons. The Cre-recombinase methodology proposed should facilitate studies on other neural lineages that express MASH-1, e.g., progenitor cells in the forebrain.
Keywords: embryonic stem cell, genetic mapping, genetic technique, method development, recombinase, regulatory gene, fusion gene, gene expression, green fluorescent protein, nucleic acid sequence, reporter gene, RNase protection assay, confocal scanning microscopy, flow cytometry, image processing, laboratory mouse, radionuclide, transgenic animal, western blotting
MOLECULAR BIOLOGY OF NEURAL CREST DEVELOPMENT
David J Anderson
California Institute Of Technology Office Of Sponsored Research, Mail Code 201-15 Pasadena, Ca 91125
Grant 5R01NS023476-03 from National Institute Of Neurological Disorders And Stroke IRG: NLS
Abstract: The cells of the neural crest migrate throughout the embryo to generate a remarkably diverse array of differentiated derivatives, including the entire peripheral nervous system. The extraordinary developmental repertoire of this apparently homogeneous precursor population is likely to require both developmental restrictions imposed by lineage, and choices among alternative fates determined by environmental interactions. The neural crest thus provides a unique biological context in which to investigate the molecular basis of the contributions of lineage and environment to developmental fate. In turn, by focusing on the expression of specific genes, we may better define lineal restriction and multipotentiality and their roles in the biology of this system. In this proposal, I describe strategies for the isolation of genes expressed specifically in neural crest cells and/or their derivatives and their use as developmental markers. We have developed procedures for the direct isolation of cDNA clones which define various crest-derived phenotypes, by differential screening of appropriate cDNA libraries. When combined with the ability to establish clonal lines from neural crest precursors, these markers may define the developmental fates which arise from different neural crest lineages and the role of environmental factors in determining the expression of these fates. In our initial experiments, we isolated several genes expressed in sympathetic neurons but not in adrenal chromaffin cells, two derivatives thought to arise from a multipotential sympathoadrenal precursor. Using these probes and established cell culture systems, we will proceed with experiments aimed at understanding how environmental factors control the expression of genes whose products define alternative phenotypes. By introducing cloned copies of these genes into various crest-derived cell types, we can examine the ways in which cell lineage determines the ability of precursors to express specific genes in response to environmental signals, and study the biochemical mechanisms underlying phenotypic plasticity. Finally, the possibility will be investigated that alterations in the chromatin structure of these genes may be used to mark latent multipotentiality and plasticity in the sympathoadrenal lineage. Ultimately, studies of these genes may permit one to create perturbations in specific cell types within the intact embryo, and examine their developmental consequences.
Keywords: GENETICS, BIOCHEMICAL GENETICS, MOLECULAR CLONING, GENETICS, DEVELOPMENTAL GENETICS, GENETICS, GENES, GENE EXPRESSION, NEUROBIOLOGY, DEVELOPMENTAL, NEUROPHYSIOLOGY, NEUROPLASTICITY, ADRENAL GLANDS, CELLS, CHROMAFFIN CELLS, GENETIC MAPPING, GENETIC MARKERS, GENETICS, BIOCHEMICAL GENETICS, GENETIC CODING, GENETICS, GENES, ONCOGENES, GENETICS, GENETIC LIBRARIES, GROWTH FACTORS (INCL. ANABOLICS), NERVE GROWTH FACTOR, NERVOUS SYSTEM AUTONOMIC, SYMPATHETIC NERVOUS SYSTEM, NERVOUS SYSTEM, PERIPHERAL NERVES, NUCLEIC ACIDS, MRNA, neurochemistry, ANIMALS, CHORDATES, BIRDS, CHICKENS, ANIMALS, CHORDATES, MAMMALS, LAGOMORPHS, ANIMALS, CHORDATES, MAMMALS, RODENTS, MYOMORPHA, MICE (LABORATORY), ANIMALS, CHORDATES, MAMMALS, RODENTS, MYOMORPHA, RATS (LABORATORY), TISSUE (CELL) CULTURE
Project start date: 1986-09-01
Project end date: 1989-08-31
MOLECULAR GENETIC DISSECTION OF CENTRAL AMYGDALA MICROCIRCUITRY UNDERLYING FEAR A
David J Anderson, Professor
California Institute Of Technology, Office Of Sponsored Research, Mail Code 201-15, Pasadena, Ca 91125
Grant 1R01MH085082-01A1 from National Institute Of Mental Health
Abstract: Psychiatric disorders, such as PTSD, depression and generalized anxiety disorder, are increasingly being recognized as dysfunctions of specific brain circuits, rather than alterations in global "brain chemistry." In order to develop new therapeutic approaches based on an understanding of underlying disease mechanisms, it is necessary to understand the normal function of the affected circuits. In this application, we propose to apply new, genetically based, techniques for manipulating neuronal function and mapping neuronal connectivity, to dissect the microcircuitry that underlies conditioned fear and its extinction. Our focus is on understanding the function of subpopulations of interneurons located in the central nucleus of the amygdala (CeA), a brain region involved in emotion. One subset of these neurons is marked by expression of protein kinase C-4 (PKC-4). Our preliminary data indicate that genetically based inactivation of these neurons enhances conditioned freezing, suggesting that these neurons may normally act to gate output from CeA. Using a recently developed genetic system for neuronal silencing, based on an ivermectin (IVM)-gated chloride channel, and an "intersectional" strategy to target expression of this heteromeric channel exclusively to PKC-4 cells in CeA, we will test this hypothesis and investigate the functional role of these neurons in fear learning and fear extinction, as well as in unconditional fear and anxiety (Specific Aim I). In Specific Aim II, we will further investigate the role of these neurons using neuronal activation strategies based on light (channelrhodopsin-2) or chemical activation. These experiments will test the necessity and sufficiency, respectively, of PKC-4 neurons in emotional behaviors mediated by the amygdala. In Specific Aim III, we will map the inputs and outputs to and from these neurons, using genetically based neuronal tracing and electrophysiological techniques. Finally, in Specific Aim IV we will test the hypothesis that activation of PKC-4 neurons is required for the behavioral effects of anxiolytic drugs, such as benzodiazepines. These studies should begin to provide a functional dissection of the amygdala at the level of granularity of specific neuronal subtypes, and may identify new cellular targets for therapeutic intervention in psychiatric disorders. Psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), exact a significant toll on public health, yet current methods to diagnose and treat them are inadequate. In order to develop a new generation of more effective treatments for these illnesses, with fewer side-effects, it is necessary to identify the underlying brain circuits that are impaired, understand the normal function of these circuits in emotional behavior, and describe how this function is altered in a given disorder. The present proposal applies an arsenal of new, genetically based, tools for dissecting neural circuit function at a level of specificity that has not previously been achieved, to understand the ´gating´ mechanisms that control the flow of information through the amygdala, a brain structure important in learning (and "unlearning") fear
Keywords: 6-namide, N-((4-hydroxy-3-methoxyphenyl)methyl)-8-methyl-, (E)-; 8-Azaspiro(4.5)decane-7, 9-dione, 8-(4-(4-(2-pyrimidinyl)-1-piperazinyl)butyl)-; 8-Methyl-N-Vanillyl-6-namide; Adverse effects; Affect; Amygdala; Amygdaloid Body; Amygdaloid Nucleus; Amygdaloid structure; Anatomic; Anatomical Sciences; Anatomy; Anterior; Anti-Anxiety Agents; Anti-Anxiety Drugs; Anxiety; Anxiolytic Agents; Anxiolytics; Behavior; Behavioral; Behavioral Assay; Benzodiazepine Compounds; Benzodiazepines; Brain; Brain Chemistry; Brain region; Buspirone; Calcium Phospholipid-Dependent Protein Kinase; Calcium-Activated Phospholipid-Dependent Kinase; Capsaicin; Cell Nucleus; Cells; Central Lateral Nucleus; Central Lateral Thalamic Nucleus; Chemicals; Chloride Channels; Complement; Complement Proteins; Connector Neuron; Cues; DISSEC; Data; Diagnosis; Disease; Disorder; Dissection; Drug effect disorder; Drugs; Dysfunction; Emotional; Emotions; Encephalon; Encephalons; Extinction; Extinction (Psychology); FOS gene; Fear; Figs; Figs - dietary; Freezing; Fright; Functional disorder; G0S7; Generalized Anxiety Disorder; Generations; Genetic; Genetic Techniques; Glutamates; Infusion; Infusion procedures; Intercalary Neuron; Intercalated Neurons; Interneurons; Internuncial Cell; Internuncial Neuron; Ion Channels, Chloride; Ivermectin; L-Glutamate; Lateral; Learning; Light; Maps; Measures; Medial; Mediating; Medication; Mental disorders; Mental health disorders; Methods; Methods and Techniques; Methods, Other; Molecular Genetic; Molecular Genetics; N-(4-(4-(2-pyrimidinyl)-1-piperazinyl)butyl)-1-cyclopentanediacetamide; Nerve Cells; Nerve Unit; Nervous System, Brain; Neural Cell; Neurocyte; Neurons; Neuroses, Post-Traumatic; Neuroses, Posttraumatic; Nucleus; Output; PKC; PTSD; Pharmaceutic Preparations; Pharmaceutical Preparations; Phospholipid-Sensitive Calcium-Dependent Protein Kinase; Photoradiation; Physiopathology; Play; Population; Post-Traumatic Stress Disorders; Prefrontal Cortex; Protein Kinase C; Protooncogene FOS; Psychiatric Disease; Psychiatric Disorder; Public Health; Role; Schizophrenia; Schizophrenic Disorders; Science of Anatomy; Serotonergic Agents; Serotonergic Drugs; Serotonin Agents; Serotonin Drugs; Specificity; Stiefel Brand of Capsaicin; Stress; Stress Disorders, Post-Traumatic; Stress Disorders, Posttraumatic; Structure; Synapses; Synaptic; System; System, LOINC Axis 4; Technics, Genetic; Techniques; Testing; Therapeutic Intervention; Tranquilizing Agents, Minor; Treatment Side Effects; Unspecified Mental Disorder; amygdaloid nuclear complex; anatomy; antianxiety agent; base; behavior test; behavioral extinction; behavioral test; c fos; c-fos Gene; c-fos Proto-Oncogenes; cellular targeting; cingulate cortex; conditioned fear; dementia praecox; depression; disease/disorder; drug action; drug/agent; effective therapy; experiment; experimental research; experimental study; fear conditioning; gain of function; improved; in vivo; intervention therapy; loss of function; mental illness; neural circuit; neural circuitry; neuronal; novel therapeutic intervention; pathophysiology; psychological disorder; public health medicine (field); public health relevance; research study; schizophrenic; side effect; social role; therapy adverse effect; tool; traumatic neurosis; treatment adverse effect; v-FOS FBJ Murine Osteosarcoma Viral Oncogene Homolog
Relevance: ITEM 7. /Relevance Psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), exact a significant toll on public health, yet current methods to diagnose and treat them are inadequate. In order to develop a new generation of more effective treatments for these illnesses, with fewer side-effects, it is necessary to identify the underlying brain circuits that are impaired, understand the normal function of these circuits in emotional behavior, and describe how this function is altered in a given disorder. The present proposal applies an arsenal of new, genetically based, tools for dissecting neural circuit function at a level of specificity that has not previously been achieved, to understand the ´gating´ mechanisms that control the flow of information through the amygdala, a brain structure important in learning (and "unlearning") fear
Project start date: 2009-06-15
Project end date: 2014-03-31
Budget start date: 15-JUN-2009
Budget end date: 31-MAR-2010
PFA/PA: PA-07-070
1R01MH085082-01A1 (2009): $403750
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